METHODS OF EVALUATING CELLS AND CELL CULTURES

Abstract

Methods of evaluating the composition of a cell culture (e.g., to distinguish chondrocytes from fibroblasts) and methods for evaluating the phenotype of an individual cell (e.g., as a chondrocyte) are disclosed. The methods may be used, for example, for assessing chondrocyte cultures used for treatment of cartilage defects. In some embodiments, the invention involves identifying cell culture composition or the identity of a cell based on expression level of a fibroblast marker. In other embodiments, the invention involves comparing expression levels of at least one chondrocyte marker and at least one fibroblast marker in a cell culture sample or in an individual cell. In illustrative embodiments, the chondrocyte marker is hyaluronan and proteoglycan link protein 1 (HAPLN1), and the fibroblast marker is microfibrillar associated protein 5 (MFAP5).

Description

[0001] This is a continuation of application Ser. No. 12/098,033, filed Apr. 4, 2008, and claims the benefit of provisional application 60/910,574 filed Apr. 6, 2007, all of which are incorporated by reference in its entirety.

[0002] This invention relates to methods of determining the composition of a cell culture, more particularly, to methods of distinguishing between chondrocytes and fibroblasts.

[0003] Injuries to articular cartilage have poor rates of repair, in part due to the lack of blood supply in cartilage tissue (Basad et al., In: Hendrich et al., Cartilage Surgery and Future Perspectives, Thieme Verlag, 49-56 (2003)). Trauma to knee joints can result in, for example, chondral and osteochondral lesions, and such injuries may progress to osteoarthritis (Brittberg et al., New England Journal of Medicine, 331(14): 889-895 (1994)). In severe cases of osteoarthritis, a total knee replacement may be needed. However, the artificial prostheses used in knee replacements have limited lifetimes, thus knee replacements are not optimal remedies, particularly for non-elderly patients (Brittberg et al., supra).

[0004] In some cases, articular cartilage injuries may be repaired by autologous chondrocyte implantation (Brittberg et al., Clin. Orthopaed. Rel. Res., 367S: S147-S155 (1999)). In this procedure, chondrocytes are harvested from a patient, expanded in cell culture to increase the number of chondrocytes, and then implanted back into the injury site of the patient. The chondrocytes are covered with a flap of periosteal tissue to seal the chondrocytes into the injury site. Although the cultured chondrocytes have a tendency to de-differentiate in culture, in a successful implant, de-differentiated chondrocytes preserve their re-differentiation potential and will re-differentiate into chondrocytes that produce a hyaline cartilaginous tissue upon implantation.

[0005] In a modified technique known as matrix-induced autologous chondrocyte implantation (MACI® implantation procedure), cultured chondrocytes are loaded onto a collagen matrix before they are implanted into the patient (Basad et al., supra). In addition, the collagen matrix can be fixed with fibrin glue rather than suturing, making it a simpler surgical technique.

[0006] Different techniques and media can be used to culture chondrocytes. Examples of serum-free media for chondrocyte culture and methods for isolation and propagation of chondrocytes are described, for example, in U.S. Pat. Nos. 6,150,163 and 7,169,610, and in U.S. Provisional Patent Application No. 60/805,307, which are incorporated herein by reference.

[0007] Fibroblasts or fibroblast-like cells (such as synoviocytes) may be co-isolated with chondrocytes and, thus, co-propagated in a cell culture in the course of preparing chondrocyte implants. Chondrocytes are known to take on a fibroblastic appearance when they de-differentiate in culture (Benya and Shaffer, Cell, 30: 215-224 (1982)). Nevertheless, they maintain their differentiation potential, i.e., they are able to re-express a chondrocytic phenotype upon implantation. As a result, it can be difficult to distinguish cultured de-differentiated chondrocytes from co-cultured fibroblasts or fibroblast-like cells based on appearance.

[0008] In addition, gene expression patterns in cultured, de-differentiated chondrocytes are different from those of native cartilage chondrocytes. For example, many markers that are highly expressed in native cartilage chondrocytes are expressed at reduced levels in cultured chondrocytes (Binette et al., J. Orthopaed. Res., 16: 207-216 (1998)). Accordingly, expression of such a chondrocyte marker may not necessarily distinguish a de-differentiated chondrocyte from cells of other types that may be present in the cell culture. Furthermore, many known fibroblast markers are expressed in both de-differentiated chondrocytes and native cartilage chondrocytes, albeit at different levels. Accordingly, the expression level of such a fibroblast marker may not necessarily indicate whether cells present in the sample are de-differentiated chondrocytes, fibroblasts, or fibroblast-like cells.

[0009] There is a need for methods of identifying chondrocytes, fibroblasts and fibroblast-like cells, particularly, methods applicable to cell culture.

[0010] In certain aspects, the methods of the invention provide methods of evaluating the composition of a cell culture (e.g., to distinguish chondrocytes from fibroblasts) and methods for evaluating the phenotype of an individual cell (e.g., as a chondrocyte). The methods of the invention may be used, for example, for assessing chondrocyte cultures used for the treatment of cartilage defects. In some embodiments, the invention involves identifying cell culture composition or the identity of a cell based on expression level of a fibroblast marker. In other embodiments, the invention involves comparing expression levels of at least one chondrocyte marker and at least one fibroblast marker in a cell culture sample or in an individual cell. In illustrative embodiments, the chondrocyte marker is hyaluronan and proteoglycan link protein 1 (HAPLN1), and the fibroblast marker is microfibrillar associated protein 5 (MFAP5).

[0011] The invention is based, at least in part, on the identification of MFAP5 as a cell phenotype marker that is highly expressed in certain non-chondrocytic cell types, such as fibroblasts and synoviocytes, while being expressed at significantly lower levels in chondrocytes. The invention is further based, at least in part, on the finding that the expression level ratios of MFAP5 and a chondrocyte marker, such as HAPLN1, is a reliable indicator of the cell phenotype in cultures derived from cartilage biopsies. While under some conditions it may be preferable to use both types of markers (i.e., fibroblast and chondrocyte markers) in order to confirm the composition of cell culture or the phenotype of an individual cell, the invention also provides embodiments in which determining the normalized expression level of the MFAP5 marker alone may be sufficient for that purpose.

[0012] In some embodiments, the fibroblast marker is other than MFAP5 and is such that its normalized expression levels are lower in chondrocytes than in fibroblasts. In some embodiments, the fibroblast marker is such that its normalized expression levels are lower in chondrocytes (e.g., primary and/or passaged chondrocytes) than in fibroblasts and/or synoviocytes. In some embodiments, the fibroblast marker is expressed at least 2-, 5-, 8-, 10-fold lower, or less, in chondrocytes than in fibroblasts and/or synoviocytes.

[0013] Thus, in one aspect, the invention provides a method of evaluating the composition of a cell culture (Method 1), e.g., cell culture that tentatively contains chondrocytes; and a method of evaluating the phenotype of an individual cell (Method 2). In the embodiments of Method 1, the expression level of a respective marker is determined as the average expression level of that marker in a plurality of cells (e.g., culture sample). In the embodiments of Method 1, the composition of a cell culture may be evaluated as a whole to determine whether it contains chondrocytes. In the embodiments of Method 2, the expression level of a marker is determined as the expression level of that marker in the individual cell being evaluated. Thus, while Method 1 identifies the composition of the cell culture, Method 2 identifies the phenotype of an individual cell, e.g., whether or not the cell is a chondrocyte.

[0014] In some embodiments, Method 1 comprises: [0015] a) obtaining a plurality of cells from a cell culture; [0016] b) determining the average expression level of a fibroblast marker of the invention in a plurality of cells from the cell culture; and [0017] c) determining the composition of the culture based on the expression level; wherein the expression level below a predetermined threshold indicates that the cell culture contains chondrocytes. Alternatively, the expression level above a predetermined threshold indicates that the cell culture does not contain chondrocytes (e.g., the culture does not comprise at least 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more chondrocytes).

[0018] In some embodiments, Method 1 involves comparing expression levels of a fibroblast marker (MFAP5 or another fibroblast marker) and a chondrocyte marker (e.g., HAPLN1) to a control or to each other. In some embodiments, the fibroblast marker and the chondrocyte marker are such that the ratio of their expression levels (chondrocyte marker to fibroblast marker) in primary and/or passaged chondrocytes is equal to, or greater than, 5, 10, 20, 30, 50, 75, 100 or more times the expression ratio in cultured fibroblasts.

[0019] In particular, in some embodiments, Method 1 comprises: [0020] a) obtaining a plurality of cells from a cell culture; [0021] b) determining the average expression level of a chondrocyte marker in the plurality of cells; [0022] c) determining the average expression level of a fibroblast marker in the plurality of cells; and [0023] d) determining the composition of the culture based on the average expression level of the chondrocyte marker and the average expression level of the fibroblast marker. In some embodiments, the culture is identified as containing chondrocytes if the expression level of the chondrocyte marker is above a predetermined threshold, while the expression level of the fibroblast marker is below a predetermined threshold. Alternatively, the expression level of the fibroblast marker above a predetermined threshold indicates that the cell culture does not contain chondrocytes (e.g., the culture does not comprise at least 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more chondrocytes).

[0024] In some embodiments, the step of determining the culture composition includes comparing the average expression levels of the chondrocyte marker and the average expression level of the fibroblast marker. In some embodiments, the markers' expression levels are compared relative to each other (thus, the thresholds may be defined, e.g., as a given difference between the expression levels of two markers, or a ratio thereof). For example, in some embodiments, a ratio of a chondrocyte marker (e.g., HAPLN1) expression level to that of a fibroblast marker (e.g., MFAP5), which is greater than a predetermined threshold, e.g., 0.25, 0.55, 1, 2, 2.2, 5, 10, 25, 50 or more, indicates that the cell culture contains chondrocytes.

[0025] In some embodiments of Method 1, the expression levels of chondrocyte and fibroblast markers are determined at the RNA level, e.g., by a standard curve method of quantitative RT-PCR or by a comparative C_T method of quantitative RT-PCR (which measures the difference in the number of threshold cycles required for the fibroblast marker and the chondrocyte markers).

[0026] In a related aspect, the invention provides a method of evaluating the phenotype of an individual cell (Method 2), e.g., using flow cytometry or single-cell RT-PCR. The method is useful for identifying individual cells from a cell culture, including a cell culture derived from cartilage or synovium, a chondrocyte culture, a fibroblast culture, synoviocyte culture, or any other appropriate culture. The method is also useful for identifying individual cells derived from any appropriate biological samples in which it is desirable to identify individual cells, including cartilage samples, synovium samples, fibroblast samples, etc. The fibroblast and chondrocyte markers in Method 2 may be chosen and evaluated as described for Method 1.

[0027] In some embodiments, Method 2 comprises [0028] a) determining the expression level of a fibroblast marker of the invention in the cell; and [0029] b) determining the phenotype of the cell based on the expression level of the fibroblast maker; wherein the expression level below a predetermined threshold indicates that the cell is a chondrocyte. Alternatively, the expression level above a predetermined threshold indicates that the cell is not a chondrocyte (e.g., a fibroblast or a synoviocyte). In some embodiments, Method 2 comprises: [0030] a) determining the expression level of a chondrocyte marker in the cell; [0031] b) determining the expression level of a fibroblast marker in the cell; and [0032] c) evaluating the phenotype of the cell based on the expression level of the chondrocyte marker and the expression level of the fibroblast marker. In some embodiments, the cell is identified as a chondrocyte if the expression level of the chondrocyte marker is above a predetermined threshold level, while the expression level of the fibroblast marker is below a predetermined threshold level. Alternatively, the cell is not a chondrocyte if the expression level of the chondrocyte marker is below a predetermined threshold level, while the expression level of the fibroblast marker is above a predetermined threshold level. In some embodiments, step c) of evaluating the phenotype of the cell includes comparing the expression levels of the chondrocyte marker and the expression level of the fibroblast marker.

[0033] Additional aspects of the invention will be set forth in the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1 is a flow diagram illustrating stages in an exemplary manufacturing process used for producing cultured chondrocytes from chondrocyte biopsies.

[0035] FIG. 2 depicts HAPLN1 expression levels in several cell strains as determined by a standard curve method of RT-PCR. Expression levels were normalized to 18S ribosomal RNA. The expression level in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly. Cell strains used are listed in Table 2.

[0036] FIG. 3 depicts expression levels of MFAP5 in the same cell strains as shown in FIG. 2, as determined by a standard curve method of RT-PCR. Expression levels were normalized to 18S ribosomal RNA. The expression level in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0037] FIG. 4 depicts the ratios of HAPLN1 and MFAP5 expression levels from FIGS. 2 and 3. The ratio in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0038] FIG. 5 depicts the ratios of HAPLN1 and MFAP5 expression levels in the same strains as shown in FIG. 2. The expression levels were determined by a comparative C_T method of RT-PCR, and the ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0039] FIG. 6 depicts HAPLN1 expression levels in a number of additional chondrocyte and synoviocyte strains. The expression levels were determined by a standard curve method of RT-PCR and normalized to 18S ribosomal RNA. The expression level in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly. Cell strains used are listed in Table 3.

[0040] FIG. 7 depicts MFAP5 expression levels in the same strains as shown in FIG. 6. The expression levels were determined by a standard curve method of RT-PCR and normalized to 18S ribosomal RNA. The expression level in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0041] FIG. 8 depicts the ratios of HAPLN1 and MFAP5 expression levels from FIGS. 6 and 7. The ratio in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0042] FIG. 9 depicts the ratios of HAPLN1 and MFAP5 expression levels in the same strains as shown in FIG. 6. The expression levels were determined by a comparative C_T method of RT-PCR, and the ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0043] FIG. 10A depicts the ratios of HAPLN1 and MFAP5 expression levels in the same strains as shown in FIGS. 2 and 6, as well as additional chondrocyte, synoviocyte, and dermal fibroblast strains identified in Table 4. The expression levels were determined by a comparative C_T method of RT-PCR using custom-designed primers and probes as described in Example 3. The HAPLN1:MFAP5 ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1). FIG. 10B depicts the ratios of HAPLN1 and MFAP5 expression levels in additional cell strains identified in Table 5. The expression levels were determined using the same methods as described for FIG. 10A.

[0044] FIG. 11 shows a comparison between expression level ratios for HAPLN1 and MFAP5 in monolayer and collagen-scaffold cultures. Cell strains used are listed in Table 7. HAPLN1 and MFAP5 expression levels were determined by a standard curve method of RT-PCR. Expression levels were normalized to 18S ribosomal RNA. The ratio in monolayer culture of primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0045] FIG. 12 shows a comparison between expression level ratios for HAPLN1 and MFAP5 in the monolayer and collagen-scaffold cultures using the same strains as shown in FIG. 11. The expression levels of HAPLN1 and MFAP5 were determined by a comparative C_T method of RT-PCR, and the ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0046] FIG. 13 depicts the change in the expression level ratios for HAPLN1 and MFAP5 as a function of culture level. Three synoviocyte strains were cultured from primary culture (culture level 1) through fourth passage (culture level 5), as shown in the figure. The expression levels of HAPLN1 and MFAP5 were determined by a comparative C_T method of RT-PCR, and the ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0047] FIGS. 14A and 14B depict the change in the expression level ratios for HAPLN1 and MFAP5 as a function of culture level. In FIGS. 14A and 14B, chondrocyte strains were sampled from cartilage (culture level 0) and then cultured from primary (culture level 1) through second passage (culture level 3), as shown in the figure. The expression levels were determined by a comparative C_T method of RT-PCR using custom-designed primers and probes as described in the Example 5. The HAPLN1:MFAP5 ratio was calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0048] FIG. 15 depicts ratios of expression levels of HAPLN1 and MFAP5 in cultures of mixed populations of chondrocytes and synoviocytes. Three trials were conducted, each with varying proportions of the two cell types. The expression levels were determined by a comparative C_T method of RT-PCR using custom-designed primers and probes as described in Example 6. The HAPLN1:MFAP5 ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0049] FIG. 16 depicts molar ratios of expression levels of HAPLN1 and MFAP5 in various cell strains, which are listed in Table 12, using absolute copy numbers of the markers as determined by an absolute quantitation method. RT-PCR was performed as described in Example 3, except that 2 μl of cDNA was used per 13 μL PCR reaction. Standard curves were prepared from synthetic HAPLN1 and MFAP5 RNA transcript standards run at 10³, 10⁴, and 10⁵ copies/reaction. The quantities of HAPLN1 and MFAP5 mRNA copies present in each test sample were determined from these standard curves.

[0050] The invention is based, at least in part, on the identification of MFAP5 as a gene that is highly expressed in certain non-chondrocytic cell types, such as fibroblasts and synoviocytes, while being expressed at significantly lower levels in chondrocytes. Accordingly, in some embodiments the invention provides methods of using MFAP5 as a cell phenotype marker. MFAP5 is a serine-threonine-rich protein that binds to fibrillins and was reported to be involved in the stabilization of type I procollagen (Lemaire et al., Arthritis & Rheumatism, 52(6): 1812-1823 (2005)). The nucleotide and amino acid sequences of human MFAP5 can be found under GenBank® Accession No. NM_--003480; its nucleotide sequence is also provided as SEQ ID NO:1. In addition to, or in place of MFAP5, other fibroblast markers can also be used in the methods of the invention, as described below.

[0051] Accordingly, in one aspect, the invention provides a method of evaluating the composition of a cell culture comprising chondrocytes (Method 1) and a method of evaluating the phenotype of an individual cell (Method 2).

[0052] In the embodiments concerning Method 1, the expression level of a respective marker is determined as the average expression level of that marker in a plurality of cells. In the embodiments of Method 1, the composition of a cell culture may be evaluated as a whole to determine whether it contains chondrocytes. In the embodiments of Method 2, the expression level of a marker is determined as the expression level of that marker in an individual cell being evaluated. Thus, while Method 1 identifies the composition of cell culture, Method 2 identifies the phenotype of an individual cell, e.g., whether or not the cell is a chondrocyte.

[0053] In some embodiments, the fibroblast marker is other than MFAP5 and is such that its normalized expression levels are lower in chondrocytes than in fibroblasts. In some embodiments, the fibroblast marker is such that its normalized expression levels are lower in chondrocytes (e.g., primary chondrocytes, cultured de-differentiated chondrocytes) than in fibroblasts (e.g. dermal fibroblasts) and/or synoviocytes. In some embodiments, the fibroblast marker is expressed at least 2-, 5-, 8-, 10-fold lower, or less, in chondrocytes than in fibroblasts and/or synoviocytes. Such additional markers can be identified using, e.g., gene array analysis, as described in, e.g., Leung et al., Trends in Genetics, 19(11): 649-659 (2003).

[0054] In some embodiments, Method 1 comprises determining the expression level of a fibroblast marker of the invention in a plurality of cells from a cell culture, wherein the expression level below a predetermined threshold indicates that the cell culture contains chondrocytes. Alternatively, the expression level above a predetermined threshold indicates that the cell culture does not contain chondrocytes (e.g., the culture does not comprise at least 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more chondrocytes).

[0055] In illustrative embodiments, the fibroblast marker is MFAP5, and a higher-than-threshold expression of MFAP5 by the cell culture indicates that the culture contains a substantial number of non-chondrocytes. In some embodiments, the predetermined threshold level is 1) equal to or less than that of MFAP5 expression in pure fibroblast cultures (e.g., 2-, 3-, 4-, or 5-fold lower) or 2) equal to or greater than (e.g., 2-, 3-, 4-, or 5-fold greater) the level of MFAP5 expression in pure chondrocyte cultures (e.g., primary chondrocytes obtained from cartilage biopsies). For fibroblast markers other than MFAP5, the predetermined threshold can be analogously selected based on the expression levels of the respective marker in pure fibroblasts and/or chondrocytes. The "predetermined" level does not need to be chosen prior to determining marker expression levels and may be chosen after expression levels are determined, based for example, on the statistical analysis of the expression results.

[0056] The plurality of cells from the culture under evaluation may be represented by a sample or an aliquot obtained from that culture. For example, in case of cultures grown on collagen matrices, punch sampling can be used as described in the Examples. The plurality of cells, typically, will contain at least the number of cells sufficient to conduct a given method of expression analysis, or more. For example, for PCR as few as 10-1,000 cells are usually sufficient, but a lower number can also be used.

[0057] In some embodiments, Method 1 and Method 2 involve comparing expression levels of a fibroblast marker (MFAP5 or another fibroblast marker) and a chondrocyte marker (e.g., HAPLN1 or another chondrocyte marker) to a control or to each other. The order in which the expression levels of either marker are determined can vary. For example, one can first determine the expression level of a chondrocyte marker and then determine the expression level of a fibroblast marker, or vice versa. In some embodiments, the expression levels of both types of markers can be determined simultaneously.

[0058] Examples of some chondrocyte markers useful in the methods of the invention, including their GenBank® accession numbers and SEQ ID NOs, are provided in Table 1. Thus, in some embodiments, the chondrocyte marker is chosen from HAPLN1, MGP, EDIL3, WISP3, AGC1, COMP, COL2A1, COL9A1, COL11A1, LECT1, S100B, CRTAC1, SOX9, and NEBL.

TABLE-US-00001 TABLE 1 Examples of chondrocyte markers GenBank ® SEQ ID Marker Name Accession No. NO Reference hyaluronan and NM_001884 SEQ ID Buckwalter et al., J. Biol. proteoglycan link NO: 2 Chem., 259(9): 5361- protein 1 5363 (1984) (HAPLN1) matrix Gla protein NM_000900 SEQ ID Monroe et al., Nat Genet., (MGP) NO: 3 21(1): 142-4 (1999) GF-like repeats NM_005711 SEQ ID Genes Dev., 12(1): 21-33 and discoidin I-like NO: 4 (1998) domains 3 (EDIL3) WNT1 inducible NM_003880 SEQ ID Kutz et al., Mol. Cell. signaling pathway NO: 5 Biol., 25(1): 414-21 protein 3 (WISP3) (2005) aggrecan 1 (AGC1) NM_001135 SEQ ID Roughley et al., Eur. Cell NO: 6 Mater., 11: 1-7 (2006) cartilage oligomeric NM_000095 SEQ ID Song et al., J. Hum. matrix protein NO: 7 Genet., 48(5): 222-5 (COMP) (2003) type II collagen NM_001844 SEQ ID Nishimura et al., Hum. (COL2A1) NO: 8 Mutat., 26(1): 36-43 (2005) type IX collagen NM_001851 SEQ ID Czarny-Ratajczak et al., (COL9A1) NO: 9 Am. J. Hum. Genet., 69(5): 969-80 (2001)) type XI collagen NM_001854 SEQ ID Poulson et al., J. Med. (COL11A1) NO: 10 Genet., 41(8):e107 (2004) leukocyte cell NM_007015 SEQ ID Hikari et al., Eur. J. derived chemotaxin NO: 11 Biochem., 260(3): 869- 1 protein (LECT1) 78 (1999) S100 calcium NM_006272 SEQ ID Steffansson et al., Nature, binding protein NO: 12 295(5844): 63-4 (1982) beta (S100B) cartilage acidic NM_018058 SEQ ID Steck et al., Biochem. J., protein 1 NO: 13 353: 169-174 (2001) (CRTAC1) SRY-box 9 protein NM_000346 SEQ ID Kou and Ikegawa, J. Biol. (SOX9) NO: 14 Chem., 279(49): 50942-8 (2004) nebulette (NEBL) NM_006393 SEQ ID Grogan et al., Arth. & NO: 15 Rheum., 56(2): 586-95 (2007)

[0059] Additional chondrocyte markers can be identified using, e.g., gene array analysis, as described in, e.g., Leung et al., Trends in Genetics, 19(11): 649-659 (2003). Generally, a chondrocyte marker is a gene or protein whose normalized expression levels are higher in chondrocytes (e.g., primary chondrocytes, cultured de-differentiated chondrocytes) than in fibroblasts (e.g. dermal fibroblasts) and/or synoviocytes. In some embodiments, the chondrocyte marker is expressed at least 2, 4, 5, 8, 10, 50, 75, 100 times or greater in chondrocytes than in fibroblasts and/or synoviocytes.

[0060] In some embodiments, the fibroblast marker and the chondrocyte marker are chosen in such a way that the ratio of their expression levels in primary chondrocytes and/or in passaged chondrocytes is equal to or greater than 5, 10, 20, 30, 50, 75, 100 or more times that in dermal fibroblasts and/or synoviocytes.

[0061] In particular, in some embodiments, Method 1 comprises: [0062] a) obtaining a plurality of cells from a cell culture; [0063] b) determining the average expression level of a chondrocyte marker in the plurality of cells; [0064] c) determining the average expression level of a fibroblast marker in the plurality of cells; and [0065] d) determining the composition of the culture based on the average expression level of the chondrocyte marker and the average expression level of the fibroblast marker. In some embodiments, the culture is identified as containing chondrocytes if the expression level of the chondrocyte marker is above a predetermined threshold, while the expression level of the fibroblast marker is below a predetermined threshold. Alternatively, the culture does not contain chondrocytes (e.g., the culture does not comprise at least 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more chondrocytes) if the expression level of the chondrocyte marker is below a predetermined threshold, while the expression level of the fibroblast marker is above a predetermined threshold level.

[0066] In further embodiments of Method 1, the invention comprises a method of evaluating the composition of a cell culture, said method comprising: [0067] a) obtaining a cartilage biopsy from a mammal; [0068] b) isolating cells from the biopsy; [0069] c) culturing cells isolated in step b) in a cell culture; [0070] d) obtaining a sample of the cell culture; [0071] e) determining the expression levels of MFAP5 and HAPLN1 in one or more cells from the sample; and [0072] f) determining the composition of the culture based on the expression levels of MFAP5 and HAPLN1.

[0073] In some embodiments, the step of determining the culture composition comprises comparing the average expression levels of the chondrocyte marker and the average expression level of the fibroblast marker. In some such embodiments, the cell culture is evaluated as containing chondrocytes when the ratio of HAPLN1 expression to that of MFAP5 is greater than 0.25. In particular embodiments, this ratio indicates that the cell culture contains at least 50% chondrocytes.

[0074] In some embodiments, the markers' expression levels are compared relative to each other (thus, the thresholds may be defined, e.g., as a given difference between the expression levels of two markers or a ratio thereof). For example, in some embodiments, a ratio of a chondrocyte marker (e.g., HAPLN1) expression level to that of a fibroblast marker (e.g., MFAP5), which is greater than a predetermined threshold, e.g., 0.25, 0.55, 1, 2, 2.2, 5, 10, 25, 50 or more, indicates that the cell culture contains chondrocytes.

[0075] In some embodiments of Method 1, the expression levels of chondrocyte and fibroblast markers are determined at the RNA level, e.g., by a standard curve method of quantitative RT-PCR or by a comparative C_T method of quantitative RT-PCR (which measures the difference in the number of threshold cycles required for the fibroblast marker and the chondrocyte markers).

[0076] In a related aspect, the invention provides a method of evaluating the phenotype of an individual cell (Method 2), e.g., using flow cytometry. The method is useful for identifying individual cells from a cell culture, including a cell culture derived from cartilage or synovium, a chondrocyte culture, a fibroblast culture, synoviocyte culture, or any other appropriate culture. The method is also useful for identifying individual cells derived from any appropriate biological samples in which it is desirable to identify individual cells, including cartilage samples, synovium samples, fibroblast samples, etc. In some embodiments, Method 2 comprises determining the expression level of a fibroblast marker of the invention in the cell, wherein the expression level below a predetermined threshold indicates that the cell is a chondrocyte. Alternatively, the expression level above a predetermined threshold indicates that the cell is not a chondrocyte (e.g., the cell is a fibroblast or a synoviocyte). In some embodiments, Method 2 comprises: [0077] a) determining the expression level of a chondrocyte marker in the cell; [0078] b) determining the expression level of a fibroblast marker in the cell; and [0079] c) evaluating the phenotype of the cell based on the expression level of the chondrocyte marker and the expression level of the fibroblast marker. In some embodiments, the cell is identified as a chondrocyte if the expression level of the chondrocyte marker is above a predetermined threshold level, while the expression level of the fibroblast marker is below a predetermined threshold level. The fibroblast and chondrocyte markers in the embodiments of Method 2 may be chosen and evaluated as described for Method 1 above.

[0080] Flow cytometry can be performed using commercially available antibodies or such antibodies may be prepared as described in, e.g., Linsenmeyer et al., Biochem. Biophys. Res. Com., 92(2): 440-6 (1980).

[0081] Cells and cultures being evaluated by the methods of this invention may be obtained from any biological sample, including any tissue, cell culture, or other material, that may or may not contain chondrocytes. In some embodiments, the cells or cultures being evaluated are of mammalian, particularly human, origin. In some embodiments, the cell culture is grown from cells released from a cartilage biopsy. For example, in autologous chondrocyte implantation, cartilage cells for the procedure are normally cultured from a cartilage biopsy of the patient receiving the implant. Carticel® autologous chondrocyte product (Genzyme Corporation, Cambridge, Mass.) is an example of a cultured chondrocyte product. In some embodiments of the invention, the cell culture comprises a collagen matrix loaded with chondrocytes. Such chondrocytes may be obtained from a cartilage biopsy and cultured prior to being loaded on the matrix, e.g., as used in the MACI® implant product. The method of the invention is useful for identifying, and/or confirming identify of cells loaded onto the collagen support prior to implanting the matrix.

[0082] To illustrate an example of the utility of the cell culture determination method, reference is made to FIG. 1. This figure illustrates the steps involved in producing a cultured chondrocyte product for autologous chondrocyte implantation, such as using Carticel® autologous chondrocytes, or for producing a cultured chondrocyte product for the MACI® implantation procedure. In step 1, a cartilage biopsy from a patient undergoing autologous chondrocyte implantation is shipped for processing (step 2). The biopsy material is digested at step 3 to release and harvest chondrocytes from the cartilage. The released cells are plated in tissue culture flasks and expanded in primary culture at step 4, and if necessary, subcultured. Once the cells reach an adequate number, they can be, optionally, cryopreserved at step 5 until the patient is ready to receive the implant. Once the patient is ready to receive the cells, they are thawed and plated into tissue culture flasks and grown to prepare an assembly culture (step 6).

[0083] For use in an autologous chondrocyte implant, if a sufficient number of cells are obtained in the assembly culture, the cells are centrifuged to a cell pellet and resuspended in shipping medium, which is the "final product" such as the Carticel® autologous chondrocyte product (step 8). This "final product" is subjected to a number of QC tests, including for example, a sterility test, a cell viability test, an endotoxin test, a mycoplasma test, and a culture composition test (step 9 "QC identity" as described herein) to ensure that the cultured cells contain a sufficient number of chondrocytes. If the cultured cells pass all QC tests, they are shipped (step 10) to the patient for implantation (step 11).

[0084] Alternatively, when the assembly culture from step 6 is to be used in a MACI® implant, the cells are resuspended in culture medium, seeded onto a collagen scaffold, and cultured for 4 days (step 7). At the end of the culture period, the cells are rinsed with shipping medium to produce a final product for MACI® implants. This product is also subjected to the QC tests outlined above. Accordingly, whether the final product is a suspension of cultured chondrocytes, such as Carticel® autologous chondrocytes, or the final product is a scaffold-seeded product for MACI® implants, the method of the invention is useful as a lot identification assay or lot release assay, to confirm the composition of a cell culture as containing chondrocytes prior to shipment of the culture. For example, the "QC identity" (step 9) can be performed at any step prior to the final product assembly, e.g., before step 4, 5, 6, 7, or 8.

[0085] Many methods of determining gene or protein expression levels are known to persons skilled in the art, e.g., as described in Sambrook et al. (eds.) Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; Current Protocols in Molecular Biology (Ausubel et al. (eds.) New York: John Wiley and Sons, 1998). Examples of such methods include polymerase chain reaction (including absolute quantitation by PCR, real time PCR(RT-PCR) and qRT-PCR, multiplex or singleplex PCR), single cell PCR, northern blot assays, nuclease protection assays, in situ hybridization assays, immunohistochemistry assays, immunocytochemistry assays, electrophoresis assays such as gel or capillary, Western blot assays, ELISAs, immunoprecipitation assays, chromatography based assays such as HPLC or gel chromotography, mass spectrometry assays, RNase protection assays, flow cytometry assays, DNA methylation assays, and histone modification analysis assays.

[0086] In all methods of the invention, expression levels, at the RNA or at the protein level, can be determined using any suitable method, including any one of conventional methods. RNA levels may be determined by, e.g., quantitative RT-PCR (e.g., TaqMan® RT-PCR or RT-PCR), Northern blotting, or any other method for determining RNA levels, or as described in the Examples. Protein levels may be determined, e.g., by using Western blotting, ELISA, flow cytometry, enzymatic activity assays, or any other method for determining protein levels. Expression levels may be scaled and/or normalized per total amount of RNA or protein in the sample and/or a control, which may typically be a housekeeping gene such, as beta-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or 18S ribosomal RNA, etc.). Normalization is typically done to account for variability in the amount of protein, DNA, or RNA input. For example, in the Examples, expression levels are normalized to 18S ribosomal RNA using a standard curve.

[0087] In illustrative embodiments, the expression levels of the fibroblast and chondrocyte markers are determined using RT-PCR, either by a standard curve or by a comparative C_T method for relative quantification. In some embodiments, absolute quantitation of marker copy numbers can be determined by preparing standard curves using known amounts of the markers. The general methods for conducting such assays are described, e.g., in Real-Time PCR Systems: Applied Biosystems 7900HT Fast Real-Time PCR System and 7300/7500 Real-Time PCR Systems, Chemistry Guide, Applied Biosystems, 2005, Part No. 4348358 Rev. E.

[0088] In the case of comparing two markers using the comparative C_T method, the amount of the ratio of expression levels of a fibroblast marker to that of a chondrocyte marker can be calculated as (1+E) (C_T,f-C_T,c), wherein C_T,f is the number of the fibroblast marker threshold cycles, C_T,c is the number of threshold cycles of the chondrocyte marker, assuming that efficiency of amplification (E) is the same for both markers and the starting amount of both markers is normalized to the same amount of endogenous control (e.g., as in two duplicate samples). In the case of E≈1, as illustrated in the Examples, the ratio can be approximated as 2 (C_T,f-C_T,c). Otherwise, the calculations can be performed as described in Appendix A of Real-Time PCR Systems: Applied Biosystems 7900HT Fast Real-Time PCR System and 7300/7500 Real-Time PCR Systems, Chemistry Guide, Applied Biosystems, 2005, Part No. 4348358 Rev. E.

[0089] Further embodiments of the invention are illustrated in the following examples, which are intended to be exemplary and not intended to be limiting on the scope of the invention.

EXAMPLES

Example 1

Expression of HAPLN1 and MFAP5 in Chondrocytes, Synoviocytes and Fibroblasts

[0090] Cell Isolation and culture--Human chondrocyte cultures were isolated from cartilage using the method for producing Carticel® autologous chondrocytes or the Protease method for producing cultured chondrocytes. Using the method for producing Carticel® autologous chondrocytes, cartilage tissue was trimmed of bone and synovium and subjected to a first digestion where tissue was enzymatically treated in collagenase solution for 18 hours at 37° C. Cells released from the first digestion were plated in tissue culture flasks with fetal bovine serum (FBS) and gentamicin containing medium (EGHXX). The cells were then subjected to a second digestion where remaining tissue from the first digestion was treated with a collagenase/trypsin solution for 2.5 hours at 37° C. Cells released from the second digestion were plated in tissue culture flasks with EGHXX. Tissue pieces remaining after the second digestion were plated in tissue culture flasks with EGHXX. Using the Protease isolation method, cartilage tissue was trimmed of bone and synovium and subjected to a first digestion in Pronase E (Sigma-Aldrich Inc., St. Louis, Mo.) solution for 1.5 hours at 37° C. The Pronase solution was then removed and a second digestion of the cartilage was performed in collagenase solution for 18 hours at 37° C. The released cells were then plated in tissue culture flasks with EGHXX. After isolation, the cell culture methods were the same for cells obtained from either isolation method. Primary cell cultures were re-fed fresh EGHXX every 2 to 4 days. When the primary culture flasks reached 50% to 80% confluence, they were trypsinized to a single cell suspension, neutralized with EGHXX to inactivate trypsin, and a cell count was performed. The resulting cell suspension was then either sampled, further expanded by subculturing, or cryopreserved for long term storage. The subculture of the primary culture is referred to as the secondary culture, or first passage. Subsequent subcultures are referred to as the second passage, third passage, fourth passage, etc. Subculturing was performed by plating cells in tissue culture flasks with EGHXX and-re-feeding with fresh EGHXX every 2 to 4 days. When the subcultures reached 80% to 100% confluence, they were trypsinized to a single cell suspension, neutralized with EGHXX to inactivate trypsin, and a cell count was performed. The resulting cell suspension was then either sampled, further expanded, or cryopreserved for long term storage.

[0091] Human synoviocyte cultures (synovium derived cell cultures, also known as synovial fibroblasts) S1 and S2 were obtained from Cell Applications Inc. (San Diego, Calif.) as cryopreserved primary cultured cells. The synoviocytes were plated in tissue culture flasks with EGHXX medium and cultured using the method for producing Carticel® autologous chondrocytes as described above. Human dermal fibroblast cultures were purchased from Cell Applications Inc. as cryopreserved-primary cultured cells. The dermal fibroblasts were cultured using the method for producing Carticel® autologous chondrocytes as described above.

[0092] The cell cultures used in this Example are listed in Table 2.

TABLE-US-00002 TABLE 2 Cell cultures used in RT-PCR Analysis (Example 1) Cell Culture Cell Type Type of Cell Culture PC Chondrocyte Primary culture C1 Chondrocyte Second Passage C2 Chondrocyte Second Passage S1 Synoviocyte Second Passage S2 Synoviocyte Second Passage F1 Dermal Fibroblast Second Passage F2 Dermal Fibroblast Second Passage

[0093] RNA and cDNA preparation--RNA was isolated from cell cultures using the TRI-spin procedure (Reno et al., Biotechniques 22: 1082-6 (1997)). Isolated RNA concentrations were determined spectrophotometrically. For the preparation of cDNA from samples PC, C1, C2, S1, S2, F1, and F2, the First Strand Synthesis Kit (Roche, Indianapolis, Ind.), using random hexamer primers was run according to the manufacturer's instructions. The resulting cDNA was stored at -20° C. or -80° C. until analysis.

[0094] Gene expression analysis--Gene expression analysis was performed using quantitative real time RT-PCR, using either a standard curve method or a comparative C_T method. The real time PCR method was based on the 5' nuclease cleavage of a dual labeled oligo probe to report sequence specific primer amplification of the target sequence ("TaqMan®" assay). Expression of genes encoding cartilage link protein (HAPLN1) and microfibrillar associated protein 5 (MFAP5) were assayed using TaqMan® Gene Expression Assays Hs00157103_m1 and Hs00185803_m1 (Applied Biosystems Inc.), respectively. The real time PCR was prepared with TaqMan® Universal PCR Master Mix, no UNG (catalog number 4324018, Applied Biosystems Inc.), appropriate TaqMan® Gene Expression Assay (Applied Biosystems) and sample cDNA were used according to the Universal PCR Mix protocol. The amplifications were run on an ABI 7500 Real-Time PCR system (Applied Biosystems Inc.) using the standard TaqMan® cycling and data collection program for this configuration. Duplicate 25 μL reactions were run with up to 5 ng of input cDNA per well. A threshold of 0.1 units was used for all assays.

[0095] Standard Curve Method--The standard curve method was performed using the Eukaryotic 18S rRNA Endogenous Control assay (catalog number 4319413E, Applied Biosystems Inc.), in which 18S rRNA is used as an internal control to normalize RT-PCR results to account for input variation. For quantitation of the relative levels of expression of each gene, dilutions of the primary chondrocyte (PC) cDNA were run to generate a standard curve with the 7500 system software. The level of each test sample's expression was determined from the standard curve, and the resulting mRNA ratios to the primary chondrocyte control (PC) were divided by the sample's 18S rRNA ratio to PC to normalize for cDNA loading.

[0096] Comparative C_T Method--Comparative C_T analysis was performed to determine the relative gene expression ratios of HAPLN1 to MFAP5 in the various samples from the real time quantitative RT-PCR gene expression analysis raw data generated as described above. The comparative Ct method provides a relative measure of the ratio of HAPLN1 to MFAP5 which allows for direct comparison between test samples without the need for standards, standard curve analysis, or actual calibrators. This method can be employed in the case of the HAPLN1 and MFAP5 assays used in this example because the following four conditions were met: 1) the assay performance was consistent from run to run; 2) equivalent amounts of RNA were run in HAPLN1, MFAP5, and endogenous control assays; 3) the C_T value for the endogenous control gene, 18S rRNA, was always lower than either the HAPLN1 C_T or MFAP5 C_T when equivalent amounts of RNA were run in each assay, and thus 18S C_T was always quantifiable when either HAPLN1 or MFAP5 were quantifiable; and 4) the method used an arbitrarily selected theoretical calibrator defined as a theoretical sample containing the ratio of HAPLN1/MFAP5 which yielded a HAPLN1 C_T value equal to the MFAP5 C_T value when the other three conditions listed above were met. The derivation of the equation used for this comparative C_T method is as follows. Where the amount of target gene in a sample, normalized to an endogenous control gene and relative to a calibrator is given by:

(1+E) (-ΔΔC_T,target gene)

wherein

E=amplification efficiency

wherein

ΔΔC_T,target gene=sample ΔC_T,target gene-calibrator ΔC_T,target gene

and wherein

ΔC_T,target gene=C_T,target gene-C_T,endogenous control gene

(See Liu, W. and Saint, D. A., Analytical Biochemistry, 302: 52-59 (2002); Livak, K. J, ABI Prism 7700 Sequence Detection System, User Bulletin 2, ABI publication 4303859, 1997). Then the ratio of HAPLN1 to MFAP5 can be described as:

(1+E) (-ΔΔC_T,HAPLN1)/(1+E) (-ΔΔC_T,MFAP5)

which equals:

(1+E) (-{[sample C_T,HAPLN1-sample C_T,endogenous control gene]-[calibrator C_T,HAPLN1-calibrator C_T,endogenous control gene]})/(1+E) (-{[sample C_T,MFAP5-sample C_T,endogenous control gene]-[calibrator C_T,MFAP5-calibrator C_T,endogenous control gene]})

If the same amount of sample is run in each assay, then the C_T of the sample endogenous control gene can be represented by the term x. If the same amount of calibrator is run in each assay, then the C_T of the calibrator endogenous control gene can be represented by the term y. Substituting these terms, the equation derives to:

(1+E) (-{[sample C_T,HAPLN1-x]-[calibrator C_T,HAPLN1-y]})/(1+E) (-{[sample C_T,MFAP5-x]-[calibrator C_T,MFAP5-y]})

which equals:

(1+E) ([x-sample C_T,HAPLN1]-[y-calibrator C_T,HAPLN1])/(1+E) ([x-sample C_T,MFAP5]-[y-calibrator C_T,MFAP5])

If the calibrator is defined as a theoretical sample containing the ratio of HAPLN1/MFAP5 which yields a C_T,HAPLN1 value equal to the C_T,MFAP5 value when equivalent amounts of calibrator are run in each assay, then the term z can be substituted for the calibrator C_T,HAPLN1 and the calibrator C_T,MFAP5. The equation then derives to:

(1+E) ([x-sample C_T,HAPLN1]-[y-z])/(1+E) ([x-sample C_T,MFAP5]-[y-z])

which equals:

(1+E) ([x-sample C_T,HAPLN1]-[y-z]-[x-sample C_T,MFAP5]-[y-z])

which equals:

(1+E) ([x-sample C_T,HAPLN1]-[x-sample C_T,MFAP5])

which equals:

(1+E) (sample C_T,MFAP5-sample C_T,HAPLN1)

And if E=1 (100% efficiency), then the relative ratio of HAPLN1 to MFAP5 equals:

2 (sample C_T,MFAP5-sample C_T,HAPLN1)

The above equations derive to a final formula leaving only two variables, the sample HAPLN1 C_T and sample MFAP5 C_T, as unknowns. This formula applies when samples are assayed under the conditions described above, and the theoretical calibrator employed is set as described above.

[0097] FIG. 2 depicts HAPLN1 expression levels in several cell strains as determined by a standard curve method of RT-PCR. FIG. 3 depicts expression levels of MFAP5 in the same cell samples as shown in FIG. 2, as determined by a standard curve method of RT-PCR. HAPLN1 was expressed at higher levels in the chondrocyte cell cultures than in the synoviocyte and fibroblast cell cultures. MFAP5 was expressed at higher levels in synoviocyte and fibroblast cell cultures than in the chondrocyte cell cultures.

[0098] FIG. 4 depicts the ratios of HAPLN1 and MFAP5 expression levels from FIGS. 2 and 3. The ratio in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0099] FIG. 5 depicts the ratios of HAPLN1 and MFAP5 expression levels in the same strains as shown in FIG. 2, however, the expression levels were determined by a comparative C_T method of RT-PCR. The results of the C_T method were similar to the results obtained by the standard curve method.

Example 2

Expression of HAPLN1 and MFAP5 in Additional Strains of Chondrocytes, Synoviocytes and Fibroblasts

[0100] Expression levels of HAPLN1 and MFAP5 were determined in additional cell cultures to confirm fidelity of the method for differentiating between chondrocyte and synoviocyte cultures. The cultures used in this Example are listed in Table 3.

TABLE-US-00003 TABLE 3 Cell cultures used in RT-PCR Analysis (Example 2) Cell Culture Cell Type Type of Cell Culture PC Chondrocyte Primary culture C3 Chondrocyte Second passage C4 Chondrocyte Second passage C5 Chondrocyte Second passage C6 Chondrocyte Second passage C7 Chondrocyte Second passage S3 Synoviocyte Second passage S4 Synoviocyte Second passage S5 Synoviocyte Second passage S6 Synoviocyte Second passage S7 Synoviocyte Second passage

[0101] Cell isolation and culture--Human chondrocyte cell cultures C3, C4, C5, C6, and C7 were isolated and cultured using the method for producing Carticel® autologous chondrocytes as described in Example 1. Human synoviocyte cultures (synovium derived cell cultures, also known as synovial fibroblasts) were either isolated at Genzyme or obtained from Cell Applications Inc. (San Diego, Calif.). Strains S4, S6, and S7 were isolated at Genzyme using various procedures. S4 was isolated by subjecting minced synovium tissue to digestion in collagenase solution for 3.5 hours at 37° C., followed by a second digestion in trypsin solution for 1 hour at 37° C. Strain S6 was isolated by subjecting minced synovium tissue to digestion in a solution containing collagenase and DNase for 2 hours at 37° C. Strain S7 was isolated by subjecting minced synovium to the method for producing Carticel® autologous chondrocytes. After isolation, the synovium derived cells were plated in tissue culture flasks with EGHXX medium and cultured using the method for producing Carticel® autologous chondrocytes as described in Example 1. Strains S3 and S5 were obtained from Cell Applications Inc. as cryopreserved first passage cells. After thawing, the cells from strains S3 and S5 were plated in tissue culture flasks with EGHXX medium and cultured using the method for producing Carticel® autologous chondrocytes as described in Example 1.

[0102] RNA isolation and cDNA preparation--RNA preparations for chondrocyte cell cultures C3, C4, C5, C6, C7 and synoviocyte cultures S3, S4, S5, S6, S7 were performed as described in Example 1. The RNA from these samples was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Inc., Foster City, Calif.) according to the manufacturer's instructions. The PC cDNA from Example 1 was used in this Example. The cDNA was stored at -20° C. or -80° C. until analysis.

[0103] Gene expression analysis--Gene expression analysis was performed using RT-PCR as described in Example 1.

[0104] FIG. 6 depicts HAPLN1 expression levels in a number of additional chondrocyte and synoviocyte strains. The expression levels were determined by a standard curve method of RT-PCR and normalized to 18S ribosomal RNA.

[0105] FIG. 7 depicts MFAP5 expression levels in the same strains as shown in FIG. 6. The expression levels were determined by a standard curve method of RT-PCR and normalized to 18S ribosomal RNA.

[0106] FIG. 8 depicts the ratios of HAPLN1 and MFAP5 expression levels from FIGS. 6 and 7. The ratio in primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0107] FIG. 9 depicts the ratios of HAPLN1 and MFAP5 expression levels in the same strains as shown in FIG. 6. The expression levels were determined by a comparative C_T method of RT-PCR, and the ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0108] The RT-PCR results in the additional cell strains were consistent with the results obtained in Example 1.

Example 3

Expression of HAPLN1 and MFAP5 in Chondrocytes, Synoviocytes and Fibroblasts Using Custom-Designed Primers and Probes

[0109] Testing of various chondrocyte, synoviocyte, and dermal fibroblast cultures was performed with primers and probes of known oligonucleotide sequences.

[0110] Cell isolation and culture--The cell strains used in this Example are listed in Tables 4 and 5 below. Human chondrocyte cell cultures C1, C2, C3, C4, C5, C6, C7, C8, C26, C28, C30, and C34 were isolated and cultured using the method for producing Carticel® autologous chondrocytes as described in Example 1. Human chondrocyte cell cultures C21, C22, C23, C24, C25, C27, C29, C31, C32, and C33 were isolated (using the Protease method) and cultured as described in Example 1. Cell isolation and culture methods for human synoviocyte cultures S1, S2, S3, S4, S5, S6, and S7 were described in Examples 1 and 2. Synoviocyte culture S9 was isolated by subjecting minced synovium tissue to digestion in a solution containing collagenase and DNase for 2 hours at 37° C. Synoviocyte culture S10 was isolated by subjecting minced synovium tissue to digestion in collagenase solution for 3.5 hours at 37° C., followed by a second digestion in trypsin solution for 1 hour at 37° C. Synoviocyte strains S11, S12, S13, S14, S15, S16, S17, and S18 were obtained from Cell Applications Inc. as cryopreserved first passage cells. Dermal fibroblast strains F1, F2, F3, F4, F5, F6, F8, F9, F10, and F11 were obtained from Cell Applications Inc. as cryopreserved primary cultured cells. All cell cultures were cultured using the method for producing Carticel® autologous chondrocytes as described in Example 1.

TABLE-US-00004 TABLE 4 First Set of Cell Cultures used in RT-PCR Analysis (Example 3) Cell Culture Cell Type Type of Cell Culture C1 Chondrocyte Second passage C2 Chondrocyte Second passage C3 Chondrocyte Second passage C4 Chondrocyte Second passage C5 Chondrocyte Second passage C6 Chondrocyte Second passage C7 Chondrocyte Second passage C8 Chondrocyte Second passage S1 Synoviocyte Second passage S2 Synoviocyte Second passage S3 Synoviocyte Second passage S4 Synoviocyte Second passage S5 Synoviocyte Second passage S6 Synoviocyte Second passage S7 Synoviocyte Second passage S9 Synoviocyte First passage S10 Synoviocyte Third passage S11 Synoviocyte Third passage S12 Synoviocyte Third passage S13 Synoviocyte Third passage F1 Dermal fibroblast Second passage F2 Dermal fibroblast Second passage F3 Dermal fibroblast Second passage F4 Dermal fibroblast Second passage F5 Dermal fibroblast Second passage F6 Dermal fibroblast Second passage

TABLE-US-00005 TABLE 5 Second Set of Cell Cultures used in RT-PCR Analysis (Example 3) Cell Culture Cell Type Type of Cell Culture C21 Chondrocyte Second Passage C22 Chondrocyte Second Passage C23 Chondrocyte Second Passage C24 Chondrocyte Second Passage C25 Chondrocyte Second Passage C26 Chondrocyte Second Passage C27 Chondrocyte Second Passage C28 Chondrocyte Second Passage C29 Chondrocyte Second Passage C30 Chondrocyte Second Passage C31 Chondrocyte Second Passage C32 Chondrocyte Second Passage C33 Chondrocyte Second Passage C34 Chondrocyte Second Passage S14 Synoviocyte Third Passage S15 Synoviocyte Third Passage S16 Synoviocyte Third Passage S17 Synoviocyte Second Passage S18 Synoviocyte Second Passage F8 Dermal fibroblast Second Passage F9 Dermal fibroblast Second Passage F10 Dermal fibroblast Second Passage F11 Dermal fibroblast Second Passage

[0111] RNA isolation and cDNA preparation--RNA preparations from chondrocyte strains C1, C2, C3, C4, C5, C6, C7, synoviocyte strains S1, S2, S3, S4, S5, S6, S7, and dermal fibroblast strains F1, and F2 were described in Examples 1 and 2. For preparation of RNA from chondrocyte strain C8, synoviocyte strains S9, S10, S11, S12, S13, S14, dermal fibroblast strains F3, F4, F5, and F6, and all strains listed in Table 5, the RNeasy® Mini Kit (Qiagen, Valencia, Calif.) RNA isolation method was used. For the RNeasy® isolation, 360 μL of lysis solution was added to cell pellets containing up to one million cells. The samples were immediately vortexed at full speed for 30 seconds, then placed at 37° C. for 5 minutes. After incubation, the samples were shaken by hand for 10 seconds, followed by another 30 second vortex at full speed. The contents of each tube were collected, and the lysate was run through a Qiashredder® column (Qiagen). Three hundred and fifty μL of the Qiashredded lysate was used in the RNeasy® procedure following the manufacturer's protocol for the isolation of RNA from animal cells. The columns were eluted with a single elution consisting of 30 μL of water. The RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Inc., Foster City, Calif.) according to the manufacturer's instructions. The resulting cDNA was stored at -20° C. or -80° C. until analysis.

[0112] Gene expression analysis--RT-PCR assays were performed with custom-designed primers and probes specific for regions of HAPLN1 and MFAP5 mRNAs. The sequence information for the custom primers and probes is shown in Table 6. Abbreviations: 6FAM=6-Carboxyfluorescein, VIC® is a trademark of Applied Biosystems Inc. and is a fluorophore, MGBNFQ=minor groove binder non-fluorescent quencher. Primers were obtained from Invitrogen Corp. (Carlsbad, Calif.). Probes were obtained from Applied Biosystems Inc. For HAPLN1, the target of the forward primer is nucleotides 543 to 570 of the HAPLN1 sequence deposited under GenBank Accession No. NM_--001884.2 (SEQ ID NO:2), the target of the reverse primer is nucleotides 603 to 622, and the target of the probe is nucleotides 584 through 601 of the same sequence. For MFAP5, the target of the forward primer is nucleotides 301 through 322 of the MFAP5 sequence deposited under Genbank Accession No. NM_--003480.2 (SEQ ID NO:1); the target of the reverse primer is nucleotides 353 through 372, and the target of the probe is nucleotides 334 through 350 of the same sequence. Real time PCR was performed with TaqMan® Fast Universal PCR Master Mix, no UNG (catalog number 4352042, Applied Biosystems Inc.), 900 nM primers, 250 nM probes, and up to 5 ng of sample cDNA, according to the TaqMan® Fast Universal PCR Mix protocol. The reaction volume was 13 μL and the amplifications were run on an ABI 7500 Real-Time PCR system (Applied Biosystems Inc.) using the default Fast TaqMan® cycling and data collection program for this configuration. A threshold of 0.1 units was used for all assays. The expression levels were determined by a comparative C_T method of RT-PCR described in Example 1.

TABLE-US-00006 TABLE 6 Custom Primer and Probe Sequences Marker Forward primer Reverse Primer Probe HAPLN1 5' TGAAGGATTAG 5' GCCCCAGTCG 5' VIC/ AAGATGATACTGTT TGGAAAGTAA 3' TACAAGGTGT GTG 3' (SEQ ID NO: GGTATTCC/ (SEQ ID NO: 16) 17) MGBNFQ 3' (SEQ ID NO: 18) MFAP5 5' CGAGGAGACGA 5' AGCGGGATCA 5' 6FAM/ TGTGACTCAAG 3' TTCACCAGAT 3' ACATTCACAG (SEQ ID NO: 19) (SEQ ID NO: AAGATCC/ 20) MGBNFQ 3' (SEQ ID NO: 21)

[0113] FIG. 10A depicts the ratios of HAPLN1 and MFAP5 expression levels in the same strains as shown in FIGS. 2 and 6, as well as additional chondrocyte, synoviocyte, and dermal fibroblast strains from Table 4. FIG. 10B depicts the ratios of HAPLN1 and MFAP5 expression levels in strains from Table 5. The results obtained with the custom-designed primers and probes were similar to the results described in Examples 1 and 2.

Example 4

Comparison of HAPLN1 and MFAP5 Expression Levels in Chondrocyte, Synoviocyte and Fibroblast Cultures in Monolayers and Collagen Scaffolds

[0114] Expression levels of HAPLN1 and MFAP5 were compared in various types of cultures in monolayers and collagen scaffolds.

[0115] Cell isolation and culture--Chondrocyte cultures C9, C10, C11, C12, C13, C14, C15, C16, C17, and C18 were isolated using the Protease method as described in Example 1, and cultured as described in Example 1. Synoviocyte culture S7 was isolated and cultured as described in Examples 1 and 2. Dermal fibroblast cultures F2 and F7 were obtained from Cell Applications Inc. as cryopreserved primary cultured cells and cultured as described in Example 1. Upon completion of second passage culture (third passage for culture S7), a sample was taken for RNA isolation (the "Day 0" or monolayer sample), and then the cells were resuspended in EGHXX medium and seeded onto a 20 cm² MAIX® scaffold (ACI-MAIX® collagen membrane, CE, Matricel GmbH, D-52134 Herzogenrath, Germany). The cells were allowed to attach for 1 hour at 37° C., then the scaffold was fed additional EGHXX and cultured for 4 days. Scaffold cultures containing synoviocytes and dermal fibroblasts were also prepared in the same manner. After 4 days of scaffold culture, the cultures were sampled using an 8 mm biopsy punch (the "Day 4" or scaffold sample), and RNA isolation was performed.

[0116] RNA Isolation and cDNA preparation--RNA was isolated using the RNeasy® Mini Kit (Qiagen, Valencia, Calif.). For the RNeasy® isolation, 360 μL of lysis solution was added to MACI® implant samples (up to two 8 mm MACI® implant punches per preparation). The samples were immediately vortexed at full speed for 30 seconds, then placed at 37° C. for 5 minutes. After incubation, the samples were shaken by hand for 10 seconds to unfold the membrane followed by another 30 second vortex at full speed. The contents of each tube were collected, and the lysate was run through a Qiashredder column (Qiagen). Three hundred and fifty μL of the Qiashredded lysate was used in the RNeasy® procedure following the manufacturer's protocol for the isolation of RNA from animal cells. The columns were eluted with a single elution consisting of 30 μL of water. Preparation of cDNA from the sample RNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Inc., Foster City, Calif.) according to the manufacturer's instructions. The cDNA was stored at -20° C. or -80° C.

[0117] Table 7 lists the cell cultures used and configurations used in this Example.

TABLE-US-00007 TABLE 7 Cell cultures used in RT-PCR Analysis (Example 4) Culture Code Cell Type Culture Type Configuration C9 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C10 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C11 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C12 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C13 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C14 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C15 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C16 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C17 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet C18 day 0 Chondrocyte Second passage 5 × 10⁵ cell pellet S7 day 0 Synoviocyte Third passage 5 × 10⁵ cell pellet F7 day 0 Dermal fibroblast Second passage 5 × 10⁵ cell pellet F2 day 0 Dermal fibroblast Second passage 5 × 10⁵ cell pellet C9 day 4 Chondrocyte MACI ® implant 8 mm punch C10 day 4 Chondrocyte MACI ® implant 8 mm punch C11 day 4 Chondrocyte MACI ® implant 8 mm punch C12 day 4 Chondrocyte MACI ® implant 8 mm punch C13 day 4 Chondrocyte MACI ® implant 8 mm punch C14 day 4 Chondrocyte MACI ® implant 8 mm punch C15 day 4 Chondrocyte MACI ® implant 8 mm punch C16 day 4 Chondrocyte MACI ® implant 8 mm punch C17 day 4 Chondrocyte MACI ® implant 8 mm punch C18 day 4 Chondrocyte MACI ® implant 8 mm punch S7 day 4 Synoviocyte MACI ® implant 8 mm punch F7 day 4 Dermal fibroblast MACI ® implant 8 mm punch F2 day 4 Dermal fibroblast MACI ® implant 8 mm punch

[0118] Gene expression analysis--Gene expression analysis of the monolayer and MACI® implant cDNA was performed in the manner outlined above in Example 1.

[0119] FIG. 11 shows a comparison between expression level ratios for HAPLN1 and MFAP5 in monolayer and collagen-scaffold cultures. HAPLN1 and MFAP5 expression levels were determined by a standard curve method of RT-PCR. Expression levels were normalized to 18S ribosomal RNA. The ratio in monolayer culture of primary chondrocytes (PC) was scaled to 1; other ratios were scaled accordingly.

[0120] FIG. 12 shows a comparison between expression level ratios for HAPLN1 and MFAP5 in the monolayer and collagen-scaffold cultures using the same strains as shown in FIG. 11. The expression levels of HAPLN1 and MFAP5 were determined by a comparative C_T method of RT-PCR, and the ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0121] The results obtained in scaffold cultures were similar to those obtained in monolayer cultures.

Example 5

Expression of HAPLN1 and MFAP5 as a Function of the Passage Number

[0122] The ratio of HAPLN1 to MFAP5 at various culture levels was investigated.

[0123] Cell isolation and culture--Chondrocyte cultures C19, C20, C31, C32, and C33 were isolated using the Protease method as described in Example 1, and cultured as described in Example 1. Synoviocyte cultures S6 and S7 were isolated and cultured as described in Examples 1 and 2. Synoviocyte culture S8 was isolated by subjecting minced synovium tissue to digestion in a solution containing collagenase and DNase for 2 hours at 37° C. Cell culture of S8 was performed as described in Example 1. For chondrocyte cultures C19, C20, C31, C32, and C33, samples of cartilage derived cells (labeled "0" in FIGS. 14A and 14B), primary cultured cells (labeled "1" in FIGS. 14A and 14B), first passage cells (labeled "2" in FIGS. 14A and 14B), and second passage cells (labeled "3" in FIGS. 14A and 14B) were taken. For synoviocyte culture S7, samples of primary cultured (labeled "1" in FIG. 13), first passage (labeled "2" in FIG. 13), second passage (labeled "3" in FIG. 13), third passage (labeled "4" in FIG. 13), and fourth passage (labeled "5" in FIG. 13) cells were taken. For synoviocyte cultures S6 and S8, samples of first passage (labeled "2" on FIG. 13), second passage (labeled "3" on FIG. 13), third passage (labeled "4" on FIG. 13), and fourth passage (labeled "5" on FIG. 13) cells were taken.

[0124] RNA isolation and cDNA preparation--RNA and cDNA were prepared using the RNeasy® Mini Kit (Qiagen) and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc.) as described in Example 3.

[0125] Gene expression analysis--Gene expression analysis of the synoviocyte samples was performed as described in Example 1. Gene expression analysis of the chondrocyte samples was performed as described in Example 3.

[0126] FIGS. 13, 14A, and 14B depict the change in the expression level ratios for HAPLN1 and MFAP5 as a function of the passage number. The expression levels were determined by a comparative C_T method of RT-PCR. The HAPLN1:MFAP5 ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1).

[0127] The ratios of HAPLN1 to MFAP5 were consistently low at all culture levels for the synoviocyte samples relative to the chondrocyte samples. The ratios of HAPLN1 to MFAP5 were consistently high at all culture levels for the chondrocyte samples relative to the synoviocyte samples.

Example 6

Expression of HAPLN1 and MFAP5 in Mixed Cell Cultures

[0128] Gene expression analysis was applied to mixed cultures of chondrocyte and synoviocyte cells to evaluate sensitivity level of the method in mixed cultures. Cell cultures of human chondrocytes and human synoviocytes were used to prepare mixtures of the two cell types at the following proportions:

[0129] 1) 0% chondrocytes/100% synoviocytes;

[0130] 2) 25% chondrocytes/75% synoviocytes;

[0131] 3) 50% chondrocytes/50% synoviocytes;

[0132] 4) 75% chondrocytes/25% synoviocytes; and

[0133] 5) 100% chondrocytes/0% synoviocytes.

[0134] Cell isolation and culture--Chondrocyte strains C5, C6, and C8 were isolated and cultured as described in Examples 1 and 2. Synoviocyte cultures S6, S7, and S9 were isolated and cultured as described in Examples 1, 2, and 3. For Mixing Experiment 1, second passage cultures of chondrocyte strain 6 (C6) and synoviocyte strain 6 (S6) were used. For Mixing Experiment 2, first passage cultures of chondrocyte strain 8 (C8) and synoviocyte strain 7 (S7) were used. For Mixing Experiment 3, first passage cultures of chondrocyte strain 5 (C5) and synoviocyte strain 9 (S9) were used.

[0135] RNA isolation and cDNA preparation--RNA and cDNA were prepared using the RNeasy® Mini Kit (Qiagen) and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc.) as described in Example 3.

[0136] Gene expression analysis--Gene expression analysis was performed as described in Example 3.

[0137] The expression levels were determined by a comparative C_T method of RT-PCR. The HAPLN1:MFAP5 ratios were calculated as 2 (C_T,MFAP5-C_T,HAPLN1). The results of the mixing experiments are provided in FIG. 15, which shows the ratios of expression levels of HAPLN1 and MFAP5 in samples consisting of mixed populations of chondrocytes and synoviocytes. In the cultures that contained 75% or less chondrocytes and 25% or more synoviocytes, the HAPLN1:MFAP5 expression level ratios were about 1 or lower. Higher ratios corresponded to higher proportions of chondrocytes in the tested samples. The ability of the assay to discern mixtures of cell cultures showed that, on average, samples composed of at least 67% chondrocytes with the balance consisting of synovial fibroblasts to produce a positive C_T,MFAP5-C_T,HAPLN1. Contamination by other cell types, such as dermal fibroblasts, can also be detected with this assay.

Example 7

Analysis of Relationship Between Assay Response and Molecular Ratio of Markers

[0138] Synthetic RNA transcripts of HAPLN1 and MFAP5 were employed to determine the relationship between the assay response and the molecular ratio of the markers in test samples. First, primary PCR (Platinum PCR Supermix, Invitrogen catalog number 11306-016) was performed on human cDNA using the primers listed in Table 8. For HAPLN1, these primers amplified nucleotides from positions 256 to 1171 of the HAPLN1 gene (Accession No. NM_--001884.2). For MFAP5, these primers amplified nucleotides from positions 32 to 728 of the MFAP5 gene (Accession No. NM_--003480.2). PCR products were analyzed on 1.5% tris-acetate EDTA (TAE) agarose gels using a 100 base pair molecular size ladder (100 bp PCR Molecular Ruler, BioRad catalog number 170-8206) for reference and SYBR Green I (Invitrogen catalog number S-7563) gel staining. The resulting primary amplicons were gel purified with 4% native tris-borate EDTA (TBE) polyacrylamide (PAGE) gels and used as a template for secondary PCR with the primers listed in Table 8. Gel-purified primary amplicon template for the secondary MFAP5 amplification was loaded at 0.56 ng per 200 μL of secondary reaction. Gel-purified primary amplicon template for the secondary HAPLN1 amplification was loaded at 2.5 ng per 900 μL of secondary reaction. Secondary amplicons were purified from native TBE PAGE gels as described and used as templates for in vitro transcription with an Ambion Megascript T7 Kit. Up to 1.3 μg of secondary amplicon template was used per 20 μL transcription reaction. The resulting transcripts were gel purified with 6% TBE-Urea (TBU) polyacrylamide gels, resuspended in 0.1 mM EDTA, and quantified spectrophotometrically with readings performed in duplicate using a conversion factor of 1.sub.A260 unit corresponding to a concentration of 40 ng/μL RNA. The gel purified transcripts were analyzed on 6% TBU gels to assess purity. After determination of purity by PAGE analysis and quantitation by spectrophotometry, the number of transcript copies per μL was determined using the conversion factors listed in Table 9. These conversion factors assume an average base molecular weight of 343 Daltons. Avagadro's constant was considered to be 6.02×10²³/mole. It was also assumed that the first base transcribed was the +1 G from the T7 promoter, followed by the target sequence. The transcripts were diluted in yeast RNA carrier buffer (20 ng/μL solution of Yeast RNA (Ambion catalog number AM7120G) in nuclease free water) at concentrations ranging from 10³ to 10⁸ copies/μL. The dilutions were then tested using the RT-PCR procedure given in Example 3 of the patent application, except that 2 μL of cDNA was used per 13 μL PCR reaction. The ratio of HAPLN1:MFAP5 was then calculated for each dilution using the comparative Ct method as described in Example 4 of the patent application.

[0139] The assay response using the copy number standards was compared to the known molecular ratio. These results are shown in Table 10. With the relationship between the assay response and the molecular ratio determined, the exact molecular ratios at various assay responses were calculated. These results, shown in Table 11, indicate that when the comparative Ct determined acceptance boundary equals 1 (for example, where HAPLN1:MFAP5=1), this corresponds to an exact molecular ratio of HAPLN1:MFAP5 of 2.212.

TABLE-US-00008 TABLE 8 Primers Used for Amplification of Copy Number Standards Reverse Primer Forward Primer Forward Primer (Primary and Marker (Primary PCR) (Secondary PCR) Secondary PCR) HAPLN1 5' GCCAAGGTGTTTT 5' TAATACGACTCACTATAGGGGC 5'CTCTGAAGCAGTA CACACAG 3' CAAGGTGTTTTCACACAG 3' GACACCA 3' (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24) MFAP5 5' CCTAGCCTGGCTT 5' TAATACGACTCACTATAGGGCCT 5'CCATTGGGTCTCT TCTTGCTC 3' AGCCTGGCTTTCTTGCTC 3' GCAAATCC 3' (SEQ ID NO: 25) (SEQ ID NO: 26) (SEQ ID NO: 27)

TABLE-US-00009 TABLE 9 Expected Transcript Sizes and Conversion Factors Expected transcript size Molecular weight Transcript (bases) (Daltons) Copies/ng HAPLN1 917 3.145 × 10⁵ g/mole 1.914 × 10⁹/ng MFAP5 698 2.394 × 10⁵ g/mole 2.515 × 10⁹/ng

TABLE-US-00010 TABLE 10 Assay Response Versus Molecular Ratio of Markers Exact molecxular Copies of Copies of ratio of Assay response, HAPLN1 MFAP5 HAPLN1:MFAP5 HAPLN1:MFAP5 1 × 10⁸ 1 × 10⁸ 1 0.470 1 × 10⁷ 1 × 10⁷ 1 0.448 1 × 10⁶ 1 × 10⁶ 1 0.423 1 × 10⁵ 1 × 10⁵ 1 0.452 1 × 10⁴ 1 × 10⁴ 1 0.443 1 × 10³ 1 × 10³ 1 0.478 Average = 0.452

TABLE-US-00011 TABLE 11 Exact Molecular Ratio of Markers at Various Assay Responses Assay Response, Exact molecular ratio of HAPLN1:MFAP5 HAPLN1:MFAP5 500 1106 100 221.2 5 11.06 1 2.212 0.2 0.4425 0.01 0.02212 0.002 0.004425

Example 8

Absolute Quantitation Analysis of Chondrocyte and Fibroblast Markers

[0140] Gene expression analysis using an absolute quantitation method was performed. Table 12 lists the cell cultures used in this Example. The various cultures were isolated and cultured as discussed in the Examples above. RNA from the cell cultures was isolated using the RNeasy Kit as discussed in Example 3. RT-PCR was performed on the cell cultures as described in Example 3, except that 2 μl of cDNA was used per 13 μL PCR reaction. In vitro transcribed RNA standards for HAPLN1 and MFAP5 (prepared as described in Example 7) were diluted to yield cDNA with final concentrations of 5×10², 5×10³, and 5×10⁴ copies per uL. Standard curves were generated by graphing the Ct results from the standards on the y-axis, versus the logarithm of the number of copies per reaction (10³, 10⁴, and 10⁵) on the x-axis. A linear trendline was fitted to the data, and the quantities of HAPLN1 and MFAP5 mRNA copies present in each test sample were determined mathematically. This method of quantitation has been previously described, e.g., in Real-Time PCR Systems: Applied Biosystems 7900HT Fast Real-Time PCR System and 7300/7500 Real-Time PCR Systems, Chemistry Guide, Applied Biosystems, 2005, Part No. 4348358 Rev. E. The molar ratio of HAPLN1:MFAP5 was then calculated for each sample. FIG. 16 depicts molar ratios of HAPLN1:MFAP5 in various cell cultures. These results indicate that the molar ratio of HAPLN1:MFAP5 in chondrocytes is high relative to synoviocytes and dermal fibroblasts.

TABLE-US-00012 TABLE 12 Cell cultures used in Absolute Quantitation Analysis (Example 8) Cell Culture Cell Type Type of Cell Culture C26 Chondrocyte Second Passage C27 Chondrocyte Second Passage C28 Chondrocyte Second Passage S14 Synoviocyte Third Passage S15 Synoviocyte Third Passage S16 Synoviocyte Third Passage F1 Dermal Fibroblast Second Passage F2 Dermal Fibroblast Second Passage

[0141] All publications and patent documents cited herein are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supersede any such material.

Sequence CWU 1

2712900DNAHomo sapiens 1attccagcct cattgtaaca cacattctac gcctagcctg gctttcttgc tctccctcat 60ctcattgttt cagcggaggc caaatctgaa gtcctttcca gggagtggct ctgttcatct 120tattcgccag ccaaagtagg aacagcgtaa gaggagagag acacattcag cagccaaagg 180actcggtgga aagagcagaa caccatagac aatatgtcgc tcttgggacc caaggtgctg 240ctgtttcttg ctgcattcat catcacctct gactggatac ccctgggggt caatagtcaa 300cgaggagacg atgtgactca agcgactcca gaaacattca cagaagatcc taatctggtg 360aatgatcccg ctacagatga aacagttttg gctgttttgg ctgatattgc accttccaca 420gatgacttgg cctccctcag tgaaaaaaat accactgcag agtgctggga tgagaaattt 480acctgcacaa ggctctactc tgtgcatcgg ccggttaaac aatgcattca tcagttatgc 540ttcaccagtt tacgacgtat gtacatcgtc aacaaggaga tctgctctcg tcttgtctgt 600aaggaacacg aagctatgaa agatgagctt tgccgtcaga tggctggtct gccccctagg 660agactccgtc gctccaatta cttccgactt cctccctgtg aaaatgtgga tttgcagaga 720cccaatggtc tgtgatcatt gaaaaagagg aaagaagaaa aaatgtatgg gtgagaggaa 780ggaggatctc cttcttctcc aaccattgac agctaaccct tagacagtat ttcttaaacc 840aatccttttg caatgtccag cttttacccc tactctctac tttttcaccc aaactgataa 900catttatctc attttctagc acttaaaata caaagtctat attattgcat aattttgctg 960cttctcaata tcatagacac agtgaataga tgatgactat atggcttata tacaaacatt 1020ctatgtacaa tttcaaggga gactaaactt taggctaata atctttacta ttgaatctgt 1080ctgatataga tcttagggtt gaagaagcta tctttgtcta tttgggctaa ccatagaatt 1140tcatttattt tcctcacaat attttcctag accaactccc catcattcac gtgttcctct 1200ttactcttac tttaactatt ttgctggctt gcccgaaaat ttgcctggca agtcttcctt 1260ataagacaca tcatggtaag ttttgtagtc ctgtaagatt ctgcaacaca gtcaagaatt 1320atacaatcct actagcaata tataaggacc caaaatgtct tctgctaagc tcagaggctg 1380gggctaaagc atgaggacta tgccagctat agaacttgga ctcataattc gctatccaat 1440ttttcatgca gttgtctagt cgggaagtaa ggttggaaac taagtctcat ttactgattc 1500gtttatgggt agtaccggga tgaacccacc accacaaagc aaattagaca acttaatgtg 1560aaatcatacc attggttgac gtttccttga gttgctactt cgttcatctt cacaacttaa 1620caagtgcacg gtcgaattat tgtgcaagtg gcttttggat atcctgattg gggcctaaga 1680agggcattca gacttgaatt ttaataggca gacagaaagt ttgcctaata gttaatacga 1740aagagtgaaa gaaacacaat attcagacaa cccacattct tatcctggct ctagcagtaa 1800ccacgtagcc ttggataagc cattttcctt cattaggtcc tggtttaatt tcctcatctt 1860taaaatgaga aggttaaatt tatcttagta ctgctgggcg cagtggctca tgcctgtaat 1920ctgagcactt tgggaagctg aggcgggtgg atcacttgag gtcagaaatt tgagacgagc 1980ctggccaaca tggtgaaacc ccatctctac taaaaataca aaaattagct gggcgtggtg 2040gcacgtgcct gtaatcccag ctactcggga ggctgaggca ggagaatcaa ttgaacctgg 2100gaggcagagg ttgcagtgag ccgagatggc gccattgcac tccagcctgg gtgacaaaag 2160caaaagtcca tcttaagaaa tatatatata tattatatat attcttagtt ctaagatttc 2220ctttaattct atgattctct ggatttaaat gcattattca tatttcttga agcttagata 2280cagtctaatt catagcaacc atatctgctt tatcctaggt gagggtagca gtccacaatg 2340gaatagaaga aaatcccatt ataacaaatg acaaattata tatcatgaat ccttctgtct 2400gactaactca ataactttct ataaaagcca atggaattca aataggagct aggagacaac 2460aagttatata tgacagtgga ggttgtattc cttttatatt gctgagaaaa ctagttaaat 2520gatcagattc ttgctgttaa gaaacaattt cgtttaatgg gatctgtaca actgatttta 2580aaaaaatgct acaaaaagcc ccaaagcata taatctctac tccttacagt ctctagaatt 2640aaatgtactc atttagacaa catattaaat gcatatttta gccactttag agaaacctca 2700taggcacaga gtttccaaga ttaattttaa gaatatcttc acgaacttga ccctcctact 2760ccacattgca acatttccat cagacagcat ttcaattcca gtattatgta tattgcaaat 2820taaacatttt aaaatatttt tttccaattt atttctcaaa ataaaatgtc ttttgttctg 2880gtaaaaaaaa aaaaaaaaaa 290021759DNAHomo sapiens 2gtgaggagaa agagcgctac gttcacttga tctccagctt ccaacttaag cagaacttga 60gagcatccga actcctggat ttcaggacaa gtgaagaaga ttctttgggc tataaagatg 120aagagtctac ttcttctggt gctgatttca atctgctggg ctgatcatct ttcagacaac 180tatactctgg atcatgacag agctattcac atccaagcag aaaatggccc ccatctactt 240gtggaagcag agcaagccaa ggtgttttca cacagaggtg gcaatgttac actgccatgt 300aaattttatc gagaccctac agcatttggc tcaggaatcc ataaaatccg aattaagtgg 360accaagctaa cttcggatta cctcaaggaa gtggatgttt ttgtttccat gggataccac 420aaaaaaacct atggaggcta ccagggtaga gtgtttctga agggaggcag tgatagtgat 480gcttctctgg tcatcacaga cctcactctg gaagattatg ggagatataa gtgtgaggtg 540attgaaggat tagaagatga tactgttgtg gtagcactgg acttacaagg tgtggtattc 600ccttactttc cacgactggg gcgctacaat ctcaattttc acgaggcgca gcaggcgtgt 660ctggaccagg atgctgtgat cgcctccttc gaccagctgt acgacgcctg gcggggcggg 720ctggactggt gcaatgccgg ctggctcagt gatggctctg tgcaatatcc catcacaaag 780cccagagagc cctgtggggg ccagaacaca gtgcccggag tcaggaacta cggattttgg 840gataaagata aaagcagata tgatgttttc tgttttacat ccaatttcaa tggccgtttt 900tactatctga tccaccccac caaactgacc tatgatgaag cggtgcaagc ttgtctcaat 960gatggtgctc agattgcaaa agtgggccag atatttgctg cctggaaaat tctcggatat 1020gaccgctgtg atgcgggctg gttggcggat ggcagcgtcc gctaccccat ctctaggcca 1080agaaggcgct gcagtcctac tgaggctgca gtgcgcttcg tgggtttccc agataaaaag 1140cataagctgt atggtgtcta ctgcttcaga gcatacaact gaatgtgccc ttagagcgca 1200tcagttttaa agtcattaag aacatgtgaa aggtgttttt tttttccaat atgaactcat 1260gcaagttacc aaaactgtga taaccctttt ttacttactg taaagagtca ttttcataag 1320atcaattcat tgatttgttt tttgtaaagc tatcattcaa tatatattat aaattaatat 1380aaatttaagg gaagctctat gtaaggagac ttagagccaa actgtttaag ctgtatcatc 1440ccaacaaagt atcctttcat gaacggggca tgcaatagct taagaattgc taggattaaa 1500ttaaggaaag taaagctact cagagcaaca ggttccacaa gcacaaactt tacacatttg 1560tacaattttg aaatgcacta caataaacaa attagagcaa cacatttgaa atacaggctt 1620ctttacataa actgagaggt tatacaaaac tcagtttcac aagggaacaa tctatacctt 1680tctaaaagtt aatatttcaa gtctctaata ggcagaatat tttactcttt aaaatcctgc 1740ctttctgacc aaaaaaaaa 17593661DNAHomo sapiens 3tggttcttat aaaaacctca cagccttcca ctaacatccc gtaggagcct ctctccctac 60tgctgctaca caagaccctg agactgacct gcaggacgaa accatgaaga gcctgatcct 120tcttgccatc ctggccgcct tagcggtagt aactttgtgt tatgaatcac atgaaagcat 180ggaatcttat gaacttaatc ccttcattaa caggagaaat gcaaatacct tcatatcccc 240tcagcagaga tggagagcta aagtccaaga gaggatccga gaacgctcta agcctgtcca 300cgagctcaat agggaagcct gtgatgacta cagactttgc gaacgctacg ccatggttta 360tggatacaat gctgcctata atcgctactt caggaagcgc cgagggacca aatgagactg 420agggaagaaa aaaaatctct ttttttctgg aggctggcac ctgattttgt atccccctgt 480agcagcatta ctgaaataca taggcttata tacaatgctt ctttcctgta tattctcttg 540tctggctgca cccctttttc ccgcccccag attgataagt aatgaaagtg cactgcagtg 600agggtcaaag gagagtcaac atatgtgatt gttccataat aaacttctgg tgtgatactt 660t 66142974DNAHomo sapiens 4agaagccccg cagccgccgc gcggagaaca gcgacagccg agcgcccggt ccgcctgtct 60gccggtgggt ctgcctgccc gcgcagcaga cccggggcgg ccgcgggagc ccgcgccccg 120cccgccgcgc ctctgccggg acccacccgc agcggagggc tgagcccgcc ggcggctccc 180cggagctcac ccacctccgc gcgccggagc gcaggcaaaa ggggaggaaa ggctcctctc 240tttagtcacc actctcgccc tctccaagaa tttgtttaac aaagcgctga ggaaagagaa 300cgtcttcttg aattctttag taggggcgga gtctgctgct gccctgcgct gccacctcgg 360ctacactgcc ctccgcgacg acccctgacc agccggggtc acgtccggga gacgggatca 420tgaagcgctc ggtagccgtc tggctcttgg tcgggctcag cctcggtgtc ccccagttcg 480gcaaaggtga tatttgtgat cccaatccat gtgaaaatgg aggtatctgt ttgccaggat 540tggctgatgg ttccttttcc tgtgagtgtc cagatggctt cacagacccc aactgttcta 600gtgttgtgga ggttgcatca gatgaagaag aaccaacttc agcaggtccc tgcactccta 660atccatgcca taatggagga acctgtgaaa taagtgaagc ataccgaggg gatacattca 720taggctatgt ttgtaaatgt ccccgaggat ttaatgggat tcactgtcag cacaacataa 780atgaatgcga agttgagcct tgcaaaaatg gtggaatatg tacagatctt gttgctaact 840attcctgtga gtgcccaggc gaatttatgg gaagaaattg tcaatacaaa tgctcaggcc 900cactgggaat tgaaggtgga attatatcaa accagcaaat cacagcttcc tctactcacc 960gagctctttt tggactccaa aaatggtatc cctactatgc acgtcttaat aagaaggggc 1020ttataaatgc gtggacagct gcagaaaatg acagatggcc gtggattcag ataaatttgc 1080aaaggaaaat gagagttact ggtgtgatta cccaaggagc caagaggatt ggaagcccag 1140agtatataaa atcctacaaa attgcctaca gtaatgatgg aaagacttgg gcaatgtaca 1200aagtgaaagg caccaatgaa gacatggtgt ttcgtggaaa cattgataac aacactccat 1260atgctaactc tttcacaccc cccataaaag ctcagtatgt aagactctat ccccaagttt 1320gtcgaagaca ttgcactttg cgaatggaac ttcttggctg tgaactgtcg ggttgttctg 1380agcctctggg tatgaaatca ggacatatac aagactatca gatcactgcc tccagcatct 1440tcagaacgct caacatggac atgttcactt gggaaccaag gaaagctcgg ctggacaagc 1500aaggcaaagt gaatgcctgg acctctggcc acaatgacca gtcacaatgg ttacaggtgg 1560atcttcttgt tccaaccaaa gtgactggca tcattacaca aggagctaaa gattttggtc 1620atgtacagtt tgttggctcc tacaaactgg cttacagcaa tgatggagaa cactggactg 1680tataccagga tgaaaagcaa agaaaagata aggttttcca gggaaatttt gacaatgaca 1740ctcacagaaa aaatgtcatc gaccctccca tctatgcacg acacataaga atccttcctt 1800ggtcctggta cgggaggatc acattgcggt cagagctgct gggctgcaca gaggaggaat 1860gaggggaggc tacatttcac aaccctcttc cctatttccc taaaagtatc tccatggaat 1920gaactgtgca aaatctgtag gaaactgaat ggtttttttt tttttttcat gaaaaagtgc 1980tcaaattatg gtaggcaact aacggtgttt ttaagggggt ctaagcctgc cttttcaatg 2040atttaatttg attttatttt atccgtcaaa tctcttaagt aacaacacat taagtgtgaa 2100ttacttttct ctcattgttt cctgaattat tcgcattggt agaaatatat tagggaaaga 2160aagtagcctt ctttttatag caagagtaaa aaagtctcaa agtcatcaaa taagagcaag 2220agttgataga gcttttacaa tcaatactca cctaattctg ataaaaggaa tactgcaatg 2280ttagcaataa gtttttttct tctgtaatga ctctacgtta tcctgtttcc ctgtgcctac 2340caaacactgt caatgtttat tacaaaattt taaagaagaa tatgtaacat gcagtactga 2400tattataatt ctcattttac tttcattatt tctaataaga gattatgtga cttctttttc 2460ttttagttct attctacatt cttaatattg tatattacct gaataattca atttttttct 2520aattgaattt cctattagtt gactaaaaga agtgtcatgt ttactcatat atgtagaaca 2580tgactgccta tcagtagatt gatctgtatt taatattcgt taattaaatc tgcagtttta 2640tttttgaagg aagccataac tatttaattt ccaaataatt gcttcataaa gaatcccata 2700ctctcagttt gcacaaaaga acaaaaaata tatatgtctc tttaaattta aatcttcatt 2760tagatggtaa ttacatatcc ttatatttac tttaaaaaat cggcttattt gtttatttta 2820taaaaaattt agcaaagaaa tattaatata gtgctgcata gtttggccaa gcatactcat 2880catttctttg ttcagctcca catttcctgt gaaactaaca tcttattgag atttgaaact 2940ggtggtagtt tcccaggaag gcacaggtgg agtt 297451307DNAHomo sapiens 5cctgagtccc gggaggaaag tgctcgccca ttcctgacct gtgacacgct cactgcgaag 60gcaggttatt agaagagtcc catgaaaggt ggctccacgg tcccagcgac atgcaggggc 120tcctcttctc cactcttctg cttgctggcc tggcacagtt ctgctgcagg gtacagggca 180ctggaccatt agatacaaca cctgaaggaa ggcctggaga agtgtcagat gcacctcagc 240gtaaacagtt ttgtcactgg ccctgcaaat gccctcagca gaagccccgt tgccctcctg 300gagtgagcct ggtgagagat ggctgtggat gctgtaaaat ctgtgccaag caaccagggg 360aaatctgcaa tgaagctgac ctctgtgacc cacacaaagg gctgtattgt gactactcag 420tagacaggcc taggtacgag actggagtgt gtgcatacct tgtagctgtt gggtgcgagt 480tcaaccaggt acattatcat aatggccaag tgtttcagcc caaccccttg ttcagctgcc 540tctgtgtgag tggggccatt ggatgcacac ctctgttcat accaaagctg gctggcagtc 600actgctctgg agctaaaggt ggaaagaagt ctgatcagtc aaactgtagc ctggaaccat 660tactacagca gctttcaaca agctacaaaa caatgccagc ttatagaaat ctcccactta 720tttggaaaaa aaaatgtctt gtgcaagcaa caaaatggac tccctgctcc agaacatgtg 780ggatgggaat atctaacagg gtgaccaatg aaaacagcaa ctgtgaaatg agaaaagaga 840aaagactgtg ttacattcag ccttgcgaca gcaatatatt aaagacaata aagattccca 900aaggaaaaac atgccaacct actttccaac tctccaaagc tgaaaaattt gtcttttctg 960gatgctcaag tactcagagt tacaaaccca ctttttgtgg aatatgcttg gataagagat 1020gctgtatccc taataagtct aaaatgatta ctattcaatt tgattgccca aatgaggggt 1080catttaaatg gaagatgctg tggattacat cttgtgtgtg tcagagaaac tgcagagaac 1140ctggagatat attttctgag ctcaagattc tgtaaaacca agcaaatggg ggaaaagtta 1200gtcaatcctg tcatataata aaaaaattag tgagtaaaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaagaaa aaaaaaaaaa aaaaaaa 130767137DNAHomo sapiens 6cggccaggtg tgtgggactg aagttcttgg agaagggagt ccaactcttc aaggtgaact 60atgaccactt tactctgggt tttcgtgact ctgagggtca tcactgcagc tgtcactgta 120gaaacttcag accatgacaa ctcgctgagt gtcagcatcc cccaaccgtc cccgctgagg 180gtcctcctgg ggacctccct caccatcccc tgctatttca tcgaccccat gcaccctgtg 240accaccgccc cttctaccgc cccactggcc ccaagaatca agtggagccg tgtgtccaag 300gagaaggagg tagtgctgct ggtggccact gaagggcgcg tgcgggtcaa cagtgcctat 360caggacaagg tctcactgcc caactacccg gccatcccca gtgacgccac cttggaagtc 420cagagcctgc gctccaatga ctctggggtc taccgctgcg aggtgatgca tggcatcgag 480gacagcgagg ccaccctgga agtcgtggtg aaaggcatcg tgttccatta cagagccatc 540tctacacgct acaccctcga ctttgacagg gcgcagcggg cctgcctgca gaacagtgcc 600atcattgcca cgcctgagca gctgcaggcc gcctacgaag acggcttcca ccagtgtgac 660gccggctggc tggctgacca gactgtcaga taccccatcc acactccccg ggaaggctgc 720tatggagaca aggatgagtt tcctggtgtg aggacgtatg gcatccgaga caccaacgag 780acctatgatg tgtactgctt cgccgaggag atggagggtg aggtctttta tgcaacatct 840ccagagaagt tcaccttcca ggaagcagcc aatgagtgcc ggcggctggg tgcccggctg 900gccaccacgg gccacgtcta cctggcctgg caggctggca tggacatgtg cagcgccggc 960tggctggccg accgcagcgt gcgctacccc atctccaagg cccggcccaa ctgcggtggc 1020aacctcctgg gcgtgaggac cgtctacgtg catgccaacc agacgggcta ccccgacccc 1080tcatcccgct acgacgccat ctgctacaca ggtgaagact ttgtggacat cccagaaaac 1140ttctttggag tggggggtga ggaggacatc accgtccaga cagtgacctg gcctgacatg 1200gagctgccac tgcctcgaaa catcactgag ggtgaagccc gaggcagcgt gatccttacc 1260gtaaagccca tcttcgaggt ctcccccagt cccctggaac ccgaggagcc cttcacgttt 1320gcccctgaaa taggggccac tgccttcgct gaggttgaga atgagactgg agaggccacc 1380aggccctggg gctttcccac acctggcctg ggccctgcca cggcattcac cagtgaggac 1440ctcgtcgtgc aggtgaccgc tgtccctggg cagccgcatt tgccaggggg ggtcgtcttc 1500cactaccgcc cgggacccac ccgctactcg ctgacctttg aggaggcaca gcaggcctgc 1560cctggcacgg gggcggtcat tgcctcgccg gagcagctcc aggccgccta cgaagcaggc 1620tatgagcagt gtgacgccgg ctggctgcgg gaccagaccg tcagataccc cattgtgagc 1680ccacggaccc catgcgtggg tgacaaggac agcagcccag gggtcaggac ctatggcgtg 1740cgcccatcaa cagagaccta cgatgtctac tgctttgtag acagacttga gggggaggtg 1800ttcttcgcca cacgccttga gcagttcacc ttccaggaag cactggagtt ctgtgaatct 1860cacaatgcca ctgccaccac gggccagctc tacgccgcct ggagccgcgg cctggacaag 1920tgctatgccg gctggctggc cgacggcagc ctccgctacc ccatcgtcac cccaaggcct 1980gcctgcggtg gggacaagcc aggcgtgaga acggtctacc tctaccctaa ccagacgggc 2040ctcccagacc cactgtcccg gcaccatgcc ttctgcttcc gaggcatttc agcggttcct 2100tctccaggag aagaagaggg tggcacaccc acatcaccct ctggtgtgga ggagtggatc 2160gtgacccaag tggttcctgg tgtggctgct gtccccgtag aagaggagac aactgctgta 2220ccctcagggg agactactgc catcctagag ttcaccaccg agccagaaaa ccagacagaa 2280tgggaaccag cctatacccc agtgggcaca tccccgctgc cagggatcct tcctacttgg 2340cctcctactg gcgccgaaac agaggaaagt acagaaggcc cttctgcaac tgaagtgccc 2400tctgcctcag aggaaccatc cccctcagag gtgccattcc cctcagagga gccatccccc 2460tcagaggaac cattcccctc agtgaggcca ttcccctcag tggagctgtt cccctcagag 2520gagccattcc cctccaagga gccatccccc tcagaggaac catcagcctc agaagagccg 2580tatacacctt caccccccga gcccagctgg actgagctgc ccagctctgg ggaggaatct 2640ggggcccctg atgtcagtgg tgacttcaca ggcagtggag atgtttcagg acaccttgac 2700ttcagtgggc agctgtcagg ggacagggca agtggactgc cctctggaga cctggactcc 2760agtggtctta cttccacagt gggctcaggc ctgactgtgg aaagtggact accctcaggg 2820gatgaagaga gaattgagtg gcccagcact cctacggttg gtgaactgcc ctctggagct 2880gagatcctag agggctctgc ctctggagtt ggggatctca gtggacttcc ttctggagaa 2940gttctagaga cctctgcctc tggagtagga gacctcagtg ggcttccttc tggagaagtt 3000ctagagacca ctgcccctgg agtagaggac atcagcgggc ttccttctgg agaagttcta 3060gagaccactg cccctggagt agaggacatc agcgggcttc cttctggaga agttctagag 3120accactgccc ctggagtaga ggacatcagc gggcttcctt ctggagaagt tctagagacc 3180actgcccctg gagtagagga catcagcggg cttccttctg gagaagttct agagaccact 3240gcccctggag tagaggacat cagcgggctt ccttctggag aagttctaga gaccgctgcc 3300cctggagtag aggacatcag cgggcttcct tctggagaag ttctagagac cgctgcccct 3360ggagtagagg acatcagcgg gcttccttct ggagaagttc tagagaccgc tgcccctgga 3420gtagaggaca tcagcgggct tccttctgga gaagttctag agaccgctgc ccctggagta 3480gaggacatca gcgggcttcc ttctggagaa gttctagaga ccgctgcccc tggagtagag 3540gacatcagcg ggcttccttc tggagaagtt ctagagaccg ctgcccctgg agtagaggac 3600atcagcgggc ttccttctgg agaagttcta gagaccgctg cccctggagt agaggacatc 3660agcgggcttc cttctggaga agttctagag actgctgccc ctggagtaga ggacatcagc 3720gggcttcctt ctggagaagt tctagagact gctgcccctg gagtagagga catcagcggg 3780cttccttctg gagaagttct agagactgct gcccctggag tagaggacat cagcgggctt 3840ccttctggag aagttctaga gactgctgcc cctggagtag aggacatcag cgggcttcct 3900tctggagaag ttctagagac tactgcccct ggagtagagg agatcagcgg gcttccttct 3960ggagaagttc tagagactac tgcccctgga gtagatgaga tcagtgggct tccttctgga 4020gaagttctag agactactgc ccctggagta gaggagatca gcgggcttcc ttctggagaa 4080gttctagaga cttctacctc tgcggtaggg gacctcagtg gacttccttc tggaggagaa 4140gttctagaga tttctgtctc tggagtagag gacatcagtg ggcttccttc tggagaggtt 4200gtagagactt ctgcctctgg aatagaggat gtcagtgaac ttccttcagg agaaggtcta 4260gagacctctg cttctggagt agaggacctc agcaggctcc cttctggaga agaagttcta 4320gagatttctg cctctggatt tggggacctc agtggagttc cttctggagg agaaggtcta 4380gagacctctg cttctgaagt agggactgac ctcagtgggc ttccttctgg aagggagggt 4440ctagagactt cagcttctgg agctgaggac ctcagtgggt tgccttctgg aaaagaagac 4500ttggtggggt cagcttctgg agacttggac ttgggcaaac tgccttctgg aactctagga 4560agtgggcaag ctccagaaac aagtggtctt ccctctggat ttagtggtga gtattctggg 4620gtggaccttg gaagtggccc accctctggc ctgcctgact ttagtggact tccatctgga 4680ttcccaactg tttccctagt ggattctaca ttggtggaag tggtcacagc ctccactgca 4740agtgaactgg aagggagggg aaccattggc atcagtggtg caggagaaat atctggactg 4800ccctccagtg agctggacat tagtgggaga gctagtggac tcccttcagg aactgaactc 4860agtggccaag catctgggtc tcctgatgtc agtggggaaa tacctggact ctttggtgtc 4920agtggacagc catcagggtt tcctgacact agtggggaaa catctggagt gactgagctt 4980agcgggctgt cctctggaca accaggtgtt agtggagaag catctggagt tctttatggc 5040actagtcaac cctttggcat aactgatctg agtggagaaa catctggggt ccctgatctc 5100agtgggcagc cttcagggtt accagggttc agtggggcaa catcaggagt ccctgacctg

5160gtttctggta ccacgagtgg cagcggtgaa tcttctggga ttacatttgt ggacaccagt 5220ttggttgaag tggcccctac tacatttaaa gaagaagaag gcttagggtc tgtggaactc 5280agtggcctcc cttccggaga ggcagatctg tcaggcaaat ctgggatggt ggatgtcagt 5340ggacagtttt ctggaacagt cgattccagt gggtttacat cccagactcc ggaattcagt 5400ggcctaccaa gtggcatagc tgaggtcagt ggagaatcct ccagagctga gattgggagc 5460agcctgccct cgggagcata ttatggcagt ggaactccat ctagtttccc cacggtctct 5520cttgtagaca gaactttggt ggaatctgta acccaggctc caacagccca agaggcagga 5580gaagggcctt ctggcatttt agaactcagt ggtgctcatt ctggagcacc agacatgtct 5640ggggagcatt ctggatttct ggacctaagt gggctgcagt ccgggctgat agagcccagc 5700ggagagccac caggtactcc atattttagt ggggattttg ccagcaccac caatgtaagt 5760ggagaatcct ctgtagccat gggcaccagt ggagaggcct caggacttcc agaagttact 5820ttaatcactt ctgagttcgt ggagggtgtt actgaaccaa ctatttctca ggaactaggc 5880caaaggcccc ctgtgacaca cacaccccag ctttttgagt ccagtggaaa agtctccaca 5940gctggggaca ttagtggagc taccccagtg ctccctgggt ctggagtaga agtatcatca 6000gtcccagaat ctagcagtga gacgtccgcc tatcctgaag ctgggttcgg ggcatctgcc 6060gcccctgagg ccagcagaga agattctggg tcccctgatc tgagtgaaac cacctctgca 6120ttccacgaag ctaaccttga gagatcctct ggcctaggag tgagcggcag cactttgaca 6180tttcaagaag gcgaggcgtc cgctgcccca gaagtgagtg gagaatccac caccaccagt 6240gatgtgggga cagaggcacc aggcttgcct tcagccactc ccacggcttc tggagacagg 6300actgaaatca gcggagacct gtctggtcac acctcgcagc tgggcgttgt catcagcacc 6360agcatcccag agtctgagtg gacccagcag acccagcgcc ctgcagagac gcatctagaa 6420attgagtcct caagcctcct gtactcagga gaagagactc acacagtcga aacagccacc 6480tccccaacag atgcttccat cccagcttct ccggaatgga aacgtgaatc agaatcaact 6540gctgcagacc aggaggtatg tgaggagggc tggaacaagt accagggcca ctgttaccgc 6600cacttcccgg accgcgagac ctgggtggat gctgagcgcc ggtgtcggga gcagcagtca 6660cacctgagca gcatcgtcac ccccgaggag caggagtttg tcaacaacaa tgcccaagac 6720taccagtgga tcggcctgaa cgacaggacc atcgaagggg acttccgctg gtcagatgga 6780caccccatgc aatttgagaa ctggcgcccc aaccagcctg acaacttttt tgccgctgga 6840gaggactgtg tggtgatgat ctggcacgag aagggcgagt ggaatgatgt tccctgcaat 6900taccacctcc ccttcacgtg taaaaagggc acagccacca cctacaaacg cagactacag 6960aagcggagct cacggcaccc tcggaggagc cgccccagca cagcccactg agaagagctt 7020ccaggacgca cccaggacgc tgagcccagg agcctgccag gctgacgtgc atcccaccca 7080gacggtgtcc tcttcttgtc gctttttgtc atataaggaa tcccattaaa aaaaaaa 713772471DNAHomo sapiens 7agaaagcgag cagccaccca gctccccgcc accgccatgg tccccgacac cgcctgcgtt 60cttctgctca ccctggctgc cctcggcgcg tccggacagg gccagagccc gttgggctca 120gacctgggcc cgcagatgct tcgggaactg caggaaacca acgcggcgct gcaggacgtg 180cgggagctgc tgcggcagca ggtcagggag atcacgttcc tgaaaaacac ggtgatggag 240tgtgacgcgt gcgggatgca gcagtcagta cgcaccggcc tacccagcgt gcggcccctg 300ctccactgcg cgcccggctt ctgcttcccc ggcgtggcct gcatccagac ggagagcggc 360gcgcgctgcg gcccctgccc cgcgggcttc acgggcaacg gctcgcactg caccgacgtc 420aacgagtgca acgcccaccc ctgcttcccc cgagtccgct gtatcaacac cagcccgggg 480ttccgctgcg aggcttgccc gccggggtac agcggcccca cccaccaggg cgtggggctg 540gctttcgcca aggccaacaa gcaggtttgc acggacatca acgagtgtga gaccgggcaa 600cataactgcg tccccaactc cgtgtgcatc aacacccggg gctccttcca gtgcggcccg 660tgccagcccg gcttcgtggg cgaccaggcg tccggctgcc agcggcgcgc acagcgcttc 720tgccccgacg gctcgcccag cgagtgccac gagcatgcag actgcgtcct agagcgcgat 780ggctcgcggt cgtgcgtgtg tgccgttggc tgggccggca acgggatcct ctgtggtcgc 840gacactgacc tagacggctt cccggacgag aagctgcgct gcccggagcg ccagtgccgt 900aaggacaact gcgtgactgt gcccaactca gggcaggagg atgtggaccg cgatggcatc 960ggagacgcct gcgatccgga tgccgacggg gacggggtcc ccaatgaaaa ggacaactgc 1020ccgctggtgc ggaacccaga ccagcgcaac acggacgagg acaagtgggg cgatgcgtgc 1080gacaactgcc ggtcccagaa gaacgacgac caaaaggaca cagaccagga cggccggggc 1140gatgcgtgcg acgacgacat cgacggcgac cggatccgca accaggccga caactgccct 1200agggtaccca actcagacca gaaggacagt gatggcgatg gtatagggga tgcctgtgac 1260aactgtcccc agaagagcaa cccggatcag gcggatgtgg accacgactt tgtgggagat 1320gcttgtgaca gcgatcaaga ccaggatgga gacggacatc aggactctcg ggacaactgt 1380cccacggtgc ctaacagtgc ccaggaggac tcagaccacg atggccaggg tgatgcctgc 1440gacgacgacg acgacaatga cggagtccct gacagtcggg acaactgccg cctggtgcct 1500aaccccggcc aggaggacgc ggacagggac ggcgtgggcg acgtgtgcca ggacgacttt 1560gatgcagaca aggtggtaga caagatcgac gtgtgtccgg agaacgctga agtcacgctc 1620accgacttca gggccttcca gacagtcgtg ctggacccgg agggtgacgc gcagattgac 1680cccaactggg tggtgctcaa ccagggaagg gagatcgtgc agacaatgaa cagcgaccca 1740ggcctggctg tgggttacac tgccttcaat ggcgtggact tcgagggcac gttccatgtg 1800aacacggtca cggatgacga ctatgcgggc ttcatctttg gctaccagga cagctccagc 1860ttctacgtgg tcatgtggaa gcagatggag caaacgtatt ggcaggcgaa ccccttccgt 1920gctgtggccg agcctggcat ccaactcaag gctgtgaagt cttccacagg ccccggggaa 1980cagctgcgga acgctctgtg gcatacagga gacacagagt cccaggtgcg gctgctgtgg 2040aaggacccgc gaaacgtggg ttggaaggac aagaagtcct atcgttggtt cctgcagcac 2100cggccccaag tgggctacat cagggtgcga ttctatgagg gccctgagct ggtggccgac 2160agcaacgtgg tcttggacac aaccatgcgg ggtggccgcc tgggggtctt ctgcttctcc 2220caggagaaca tcatctgggc caacctgcgt taccgctgca atgacaccat cccagaggac 2280tatgagaccc atcagctgcg gcaagcctag ggaccagggt gaggacccgc cggatgacag 2340ccaccctcac cgcggctgga tgggggctct gcacccagcc ccaaggggtg gccgtcctga 2400gggggaagtg agaagggctc agagaggaca aaataaagtg tgtgtgcagg gaaaaaaaaa 2460aaaaaaaaaa a 247185087DNAHomo sapiens 8aacgggcgcc gcggcgggga gaagacgcag agcgctgctg ggctgccggg tctcccgctt 60ccccctcctg ctccaagggc ctcctgcatg agggcgcggt agagacccgg acccgcgccg 120tgctcctgcc gtttcgctgc gctccgcccg ggcccggctc agccaggccc cgcggtgagc 180catgattcgc ctcggggctc cccagacgct ggtgctgctg acgctgctcg tcgccgctgt 240ccttcggtgt cagggccagg atgtccagga ggctggcagc tgtgtgcagg atgggcagag 300gtataatgat aaggatgtgt ggaagccgga gccctgccgg atctgtgtct gtgacactgg 360gactgtcctc tgcgacgaca taatctgtga agacgtgaaa gactgcctca gccctgagat 420ccccttcgga gagtgctgcc ccatctgccc aactgacctc gccactgcca gtgggcaacc 480aggaccaaag ggacagaaag gagaacctgg agacatcaag gatattgtag gacccaaagg 540acctcctggg cctcagggac ctgcagggga acaaggaccc agaggggatc gtggtgacaa 600aggtgaaaaa ggtgcccctg gacctcgtgg cagagatgga gaacctggga cccctggaaa 660tcctggcccc cctggtcctc ccggcccccc tggtccccct ggtcttggtg gaaactttgc 720tgcccagatg gctggaggat ttgatgaaaa ggctggtggc gcccagttgg gagtaatgca 780aggaccaatg ggccccatgg gacctcgagg acctccaggc cctgcaggtg ctcctgggcc 840tcaaggattt caaggcaatc ctggtgaacc tggtgaacct ggtgtctctg gtcccatggg 900tccccgtggt cctcctggtc cccctggaaa gcctggtgat gatggtgaag ctggaaaacc 960tggaaaagct ggtgaaaggg gtccgcctgg tcctcagggt gctcgtggtt tcccaggaac 1020cccaggcctt cctggtgtca aaggtcacag aggttatcca ggcctggacg gtgctaaggg 1080agaggcgggt gctcctggtg tgaagggtga gagtggttcc ccgggtgaga acggatctcc 1140gggcccaatg ggtcctcgtg gcctgcctgg tgaaagagga cggactggcc ctgctggcgc 1200tgcgggtgcc cgaggcaacg atggtcagcc aggccccgca gggcctccgg gtcctgtcgg 1260tcctgctggt ggtcctggct tccctggtgc tcctggagcc aagggtgaag ccggccccac 1320tggtgcccgt ggtcctgaag gtgctcaagg tcctcgcggt gaacctggta ctcctgggtc 1380ccctgggcct gctggtgcct ccggtaaccc tggaacagat ggaattcctg gagccaaagg 1440atctgctggt gctcctggca ttgctggtgc tcctggcttc cctgggccac ggggccctcc 1500tggccctcaa ggtgcaactg gtcctctggg cccgaaaggt cagacgggtg aacctggtat 1560tgctggcttc aaaggtgaac aaggccccaa gggagaacct ggccctgctg gcccccaggg 1620agcccctgga cccgctggtg aagaaggcaa gagaggtgcc cgtggagagc ctggtggcgt 1680tgggcccatc ggtccccctg gagaaagagg tgctcccggc aaccgcggtt tcccaggtca 1740agatggtctg gcaggtccca agggagcccc tggagagcga gggcccagtg gtcttgctgg 1800ccccaaggga gccaacggtg accctggccg tcctggagaa cctggccttc ctggagcccg 1860gggtctcact ggccgccctg gtgatgctgg tcctcaaggc aaagttggcc cttctggagc 1920ccctggtgaa gatggtcgtc ctggacctcc aggtcctcag ggggctcgtg ggcagcctgg 1980tgtcatgggt ttccctggcc ccaaaggtgc caacggtgag cctggcaaag ctggtgagaa 2040gggactgcct ggtgctcctg gtctgagggg tcttcctggc aaagatggtg agacaggtgc 2100tgcaggaccc cctggccctg ctggacctgc tggtgaacga ggcgagcagg gtgctcctgg 2160gccatctggg ttccagggac ttcctggccc tcctggtccc ccaggtgaag gtggaaaacc 2220aggtgaccag ggtgttcccg gtgaagctgg agcccctggc ctcgtgggtc ccaggggtga 2280acgaggtttc ccaggtgaac gtggctctcc cggtgcccag ggcctccagg gtccccgtgg 2340cctccccggc actcctggca ctgatggtcc caaaggtgca tctggcccag caggcccccc 2400tggggctcag ggccctccag gtcttcaggg aatgcctggc gagaggggag cagctggtat 2460cgctgggccc aaaggcgaca ggggtgacgt tggtgagaaa ggccctgagg gagcccctgg 2520aaaggatggt ggacgaggcc tgacaggtcc cattggcccc cctggcccag ctggtgctaa 2580tggcgagaag ggagaagttg gacctcctgg tcctgcagga agtgctggtg ctcgtggcgc 2640tccgggtgaa cgtggagaga ctgggccccc cggaccagcg ggatttgctg ggcctcctgg 2700tgctgatggc cagcctgggg ccaagggtga gcaaggagag gccggccaga aaggcgatgc 2760tggtgcccct ggtcctcagg gcccctctgg agcacctggg cctcagggtc ctactggagt 2820gactggtcct aaaggagccc gaggtgccca aggccccccg ggagccactg gattccctgg 2880agctgctggc cgcgttggac ccccaggctc caatggcaac cctggacccc ctggtccccc 2940tggtccttct ggaaaagatg gtcccaaagg tgctcgagga gacagcggcc cccctggccg 3000agctggtgaa cccggcctcc aaggtcctgc tggaccccct ggcgagaagg gagagcctgg 3060agatgacggt ccctctggtg ccgaaggtcc accaggtccc cagggtctgg ctggtcagag 3120aggcatcgtc ggtctgcctg ggcaacgtgg tgagagagga ttccctggct tgcctggccc 3180gtcgggtgag cccggcaagc agggtgctcc tggagcatct ggagacagag gtcctcctgg 3240ccccgtgggt cctcctggcc tgacgggtcc tgcaggtgaa cctggacgag agggaagccc 3300cggtgctgat ggcccccctg gcagagatgg cgctgctgga gtcaagggtg atcgtggtga 3360gactggtgct gtgggagctc ctggagcccc tgggccccct ggctcccctg gccccgctgg 3420tccaactggc aagcaaggag acagaggaga agctggtgca caaggcccca tgggaccctc 3480aggaccagct ggagcccggg gaatccaggg tcctcaaggc cccagaggtg acaaaggaga 3540ggctggagag cctggcgaga gaggcctgaa gggacaccgt ggcttcactg gtctgcaggg 3600tctgcccggc cctcctggtc cttctggaga ccaaggtgct tctggtcctg ctggtccttc 3660tggccctaga ggtcctcctg gccccgtcgg tccctctggc aaagatggtg ctaatggaat 3720ccctggcccc attgggcctc ctggtccccg tggacgatca ggcgaaaccg gccctgctgg 3780tcctcctgga aatcctggac cccctggtcc tccaggtccc cctggccctg gcatcgacat 3840gtccgccttt gctggcttag gcccgagaga gaagggcccc gaccccctgc agtacatgcg 3900ggccgaccag gcagccggtg gcctgagaca gcatgacgcc gaggtggatg ccacactcaa 3960gtccctcaac aaccagattg agagcatccg cagccccgag ggctcccgca agaaccctgc 4020tcgcacctgc agagacctga aactctgcca ccctgagtgg aagagtggag actactggat 4080tgaccccaac caaggctgca ccttggacgc catgaaggtt ttctgcaaca tggagactgg 4140cgagacttgc gtctacccca atccagcaaa cgttcccaag aagaactggt ggagcagcaa 4200gagcaaggag aagaaacaca tctggtttgg agaaaccatc aatggtggct tccatttcag 4260ctatggagat gacaatctgg ctcccaacac tgccaacgtc cagatgacct tcctacgcct 4320gctgtccacg gaaggctccc agaacatcac ctaccactgc aagaacagca ttgcctatct 4380ggacgaagca gctggcaacc tcaagaaggc cctgctcatc cagggctcca atgacgtgga 4440gatccgggca gagggcaata gcaggttcac gtacactgcc ctgaaggatg gctgcacgaa 4500acataccggt aagtggggca agactgttat cgagtaccgg tcacagaaga cctcacgcct 4560ccccatcatt gacattgcac ccatggacat aggagggccc gagcaggaat tcggtgtgga 4620catagggccg gtctgcttct tgtaaaaacc tgaacccaga aacaacacaa tccgttgcaa 4680acccaaagga cccaagtact ttccaatctc agtcactcta ggactctgca ctgaatggct 4740gacctgacct gatgtccatt catcccaccc tctcacagtt cggacttttc tcccctctct 4800ttctaagaga cctgaactgg gcagactgca aaataaaatc tcggtgttct atttatttat 4860tgtcttcctg taagaccttc gggtcaaggc agaggcagga aactaactgg tgtgagtcaa 4920atgccccctg agtgactgcc cccagcccag gccagaagac ctcccttcag gtgccgggcg 4980caggaactgt gtgtgtccta cacaatggtg ctattctgtg tcaaacacct ctgtattttt 5040taaaacatca attgatatta aaaatgaaaa gattattgga aagtaca 508793704DNAHomo sapiens 9ccctcaccgg gggcaggagg gaccaaggct gggcccagaa cacatagtcc tagggtaaca 60gtgaaggggt cgtgagggga cagtgactcc cttccaaccc cttcttcata gggactgttg 120gcaaacaaag aaaatcaact gggaaaatga agacctgctg gaaaattcca gttttcttct 180ttgtgtgcag tttcctggaa ccctgggcat ctgcagctgt caagcgtcgc cccagattcc 240ctgtcaattc caattctaat ggtggaaatg aactctgtcc aaagatcagg attggccaag 300atgacttacc agggtttgat ctgatctctc agttccaggt agataaagca gcatctagaa 360gagctatcca gagagtagtg ggatcagcta cattgcaggt ggcttacaag ttgggaaata 420atgtagactt caggattcca actaggaatt tatatcccag tggactgcct gaagaatact 480ccttcttgac gacgtttcga atgactggaa gcactctcaa aaagaactgg aacatttggc 540agattcagga ttcctctggg aaggagcaag ttggcataaa gattaatggc caaacacaat 600ctgttgtatt ttcatacaag ggactggatg gaagtctcca aacagcagcc ttttcgaatt 660tgtcctcctt gtttgattcc cagtggcata agatcatgat tggcgtggag aggagtagtg 720ctactctttt tgttgactgc aacaggattg aatctttacc tataaagcca agaggcccaa 780ttgacattga tggctttgct gtgctgggaa aacttgcaga taatcctcaa gtttctgttc 840catttgaact tcaatggatg ctgatccatt gtgaccccct gcggcccagg agagaaactt 900gccatgagct gccagccaga ataacgccca gccagaccac cgacgagaga ggtcccccgg 960gtgagcaggg tcctcccggg cctccgggcc cccctggagt tccaggcatc gatggcatcg 1020acggtgaccg aggtcctaag ggccccccgg gccccccggg tcctgcaggt gaaccgggaa 1080agccaggagc tccaggcaag cctggcacac ctggcgctga tggattaaca ggacctgatg 1140gatcccctgg ctccattggg tcaaagggac aaaaaggaga acctggtgtg cctggatcgc 1200gtggatttcc aggccgtggt attcctggac cccctggtcc tcctgggaca gcaggactcc 1260ctggagagct tggccgtgta ggacctgttg gtgaccctgg gagaagagga ccacctggcc 1320cccctggccc cccaggaccc agaggaacaa ttggctttca tgatggagat ccattgtgtc 1380ccaatgcctg tccaccaggt cgctcaggat atccaggcct accaggcatg aggggtcata 1440aaggggctaa aggagaaatt ggtgaaccag gaagacaagg acacaagggt gaagaaggtg 1500accagggaga actcggagaa gttggagctc aaggacctcc aggagcccag ggtttgcgag 1560gcatcaccgg catagttggg gacaaagggg aaaaaggtgc tcggggctta gatggtgaac 1620ctgggcctca gggtcttcct ggtgcacctg gtgatcaagg acagcgagga cctccaggag 1680aagcaggtcc caaaggagat agaggggctg aaggtgctag aggaattcct ggtctccctg 1740ggcccaaagg agacacgggt ttgccaggtg tggatggccg tgatgggatc cctggaatgc 1800ctggaacaaa gggtgaacca ggaaaacctg ggcctcctgg tgatgcagga ttgcaggggt 1860taccaggtgt acctggaatt cctggtgcaa agggtgttgc tggtgaaaag ggtagcacag 1920gtgctccagg gaagcctggt cagatgggaa attcaggcaa accgggccaa caggggcctc 1980caggagaggt gggaccccga ggaccccagg ggcttcctgg cagtagagga gaattaggac 2040cagtgggatc cccaggccta ccaggtaaac tgggttctct gggtagccct ggcctccctg 2100gcttgcctgg gccccctgga cttcctggaa tgaaaggtga caggggtgta gtcggtgaac 2160cgggtccaaa gggtgaacag ggtgcctctg gtgaagaagg tgaagcagga gaaagggggg 2220aacttggaga tataggatta cctggcccaa agggatctgc aggtaatcct ggggaacctg 2280gcttgagagg gcctgaggga agtcgggggc ttcctggagt ggaaggacca agaggaccac 2340ctggaccccg gggtgtgcag ggagaacagg gtgccaccgg cctgcctggt gtccagggcc 2400ctccgggtag agcaccgaca gatcagcaca ttaagcaggt ttgcatgaga gtcatacaag 2460aacattttgc tgagatggct gccagtctta agcgtccaga ctcaggtgcc actgggcttc 2520ctggaaggcc tggccctcct ggtccccccg gccctcctgg agagaatggt ttcccaggcc 2580agatgggaat tcgtggcctt ccgggcatta aggggccccc tggtgctctt ggtttgaggg 2640gacctaaagg tgacttggga gaaaaggggg agcgtggccc tccaggaaga ggtcccaacg 2700gtttgcctgg agctataggt ctcccaggtg acccaggccc tgccagctat ggcagaaatg 2760gccgagacgg tgagcgaggc cccccagggg tggcaggaat tcctggagtg cctggacccc 2820cgggacctcc tgggcttccc ggtttctgtg agccagcctc ctgcaccatg caggctggtc 2880agcgagcatt taacaaaggg cctgaccctt gaaaggctta ctgctgcatg gctgtctgca 2940tgaaccacgc ctggtgaagg agcctgggtg agaaacacca tccaaagctg gggcaaagat 3000gattaccttc agcatgatta caatgtatta ccttcagtat gattacagaa gtcctacttg 3060acaatcacat atagaagaac ggtgctattc agtaagttct ctttcctttc ccttggaggg 3120aagacagcag agtcatcagt taaaaaaaaa aaaagaaaac caaacacctc ccttgaataa 3180atttatactc ctgttcccag gatcttgagc tttagtgtgc tatacctatg tgtcttatcg 3240tgggccactg tgccaataaa caaaaacaac tgtttggttt acctcagttg cagtagttat 3300tttcatttag aagttgttct cagattattg tttcagttat atagaggatt actagactag 3360ttatgaagaa accccactac attcaatgga attggtgctt aaaatctcat cgatgtgctg 3420tctctggagt gataagaaag ggctacatct cccgaaatga tttctttacg tcatgtattg 3480gtttccttct tcaccttgaa cttttgttga actgtatgta ctttacccca aacctgttaa 3540tattttgagc gcttctatgt gaaagcaaag aaataatttt aatactctgg cattcataaa 3600ttttattgat gagattattt attttaaagg tttgaggtaa catctctggt tgtaccaaag 3660aagaaataaa tatggtttct taatctcttg catgttttct tata 3704107291DNAHomo sapiens 10acacagtact ctcagcttgt tggtggaagc ccctcatctg ccttcattct gaaggcaggg 60cccggcagag gaaggatcag agggtcgcgg ccggagggtc ccggccggtg gggccaactc 120agagggagag gaaagggcta gagacacgaa gaacgcaaac catcaaattt agaagaaaaa 180gccctttgac tttttccccc tctccctccc caatggctgt gtagcaaaca tccctggcga 240taccttggaa aggacgaagt tggtctgcag tcgcaatttc gtgggttgag ttcacagttg 300tgagtgcggg gctcggagat ggagccgtgg tcctctaggt ggaaaacgaa acggtggctc 360tgggatttca ccgtaacaac cctcgcattg accttcctct tccaagctag agaggtcaga 420ggagctgctc cagttgatgt actaaaagca ctagattttc acaattctcc agagggaata 480tcaaaaacaa cgggattttg cacaaacaga aagaattcta aaggctcaga tactgcttac 540agagtttcaa agcaagcaca actcagtgcc ccaacaaaac agttatttcc aggtggaact 600ttcccagaag acttttcaat actatttaca gtaaaaccaa aaaaaggaat tcagtctttc 660cttttatcta tatataatga gcatggtatt cagcaaattg gtgttgaggt tgggagatca 720cctgtttttc tgtttgaaga ccacactgga aaacctgccc cagaagacta tcccctcttc 780agaactgtta acatcgctga cgggaagtgg catcgggtag caatcagcgt ggagaagaaa 840actgtgacaa tgattgttga ttgtaagaag aaaaccacga aaccacttga tagaagtgag 900agagcaattg ttgataccaa tggaatcacg gtttttggaa caaggatttt ggatgaagaa 960gtttttgagg gggacattca gcagtttttg atcacaggtg atcccaaggc agcatatgac 1020tactgtgagc attatagtcc agactgtgac tcttcagcac ccaaggctgc tcaagctcag 1080gaacctcaga tagatgagta tgcaccagag gatataatcg aatatgacta tgagtatggg 1140gaagcagagt ataaagaggc tgaaagtgta acagagggac ccactgtaac tgaggagaca 1200atagcacaga cggaggcaaa catcgttgat gattttcaag aatacaacta tggaacaatg 1260gaaagttacc agacagaagc tcctaggcat gtttctggga caaatgagcc aaatccagtt 1320gaagaaatat ttactgaaga atatctaacg ggagaggatt atgattccca gaggaaaaat 1380tctgaggata cactatatga aaacaaagaa atagacggca gggattctga tcttctggta 1440gatggagatt taggcgaata tgatttttat gaatataaag aatatgaaga taaaccaaca 1500agccccccta atgaagaatt tggtccaggt gtaccagcag aaactgatat tacagaaaca 1560agcataaatg gccatggtgc atatggagag aaaggacaga aaggagaacc agcagtggtt 1620gagcctggta tgcttgtcga aggaccacca ggaccagcag

gacctgcagg tattatgggt 1680cctccaggtc tacaaggccc cactggaccc cctggtgacc ctggcgatag gggcccccca 1740ggacgtcctg gcttaccagg ggctgatggt ctacctggtc ctcctggtac tatgttgatg 1800ttaccgttcc gttatggtgg tgatggttcc aaaggaccaa ccatctctgc tcaggaagct 1860caggctcaag ctattcttca gcaggctcgg attgctctga gaggcccacc tggcccaatg 1920ggtctaactg gaagaccagg tcctgtgggg gggcctggtt catctggggc caaaggtgag 1980agtggtgatc caggtcctca gggccctcga ggcgtccagg gtccccctgg tccaacggga 2040aaacctggaa aaaggggtcg tccaggtgca gatggaggaa gaggaatgcc aggagaacct 2100ggggcaaagg gagatcgagg gtttgatgga cttccgggtc tgccaggtga caaaggtcac 2160aggggtgaac gaggtcctca aggtcctcca ggtcctcctg gtgatgatgg aatgagggga 2220gaagatggag aaattggacc aagaggtctt ccaggtgaag ctggcccacg aggtttgctg 2280ggtccaaggg gaactccagg agctccaggg cagcctggta tggcaggtgt agatggcccc 2340ccaggaccaa aagggaacat gggtccccaa ggggagcctg ggcctccagg tcaacaaggg 2400aatccaggac ctcagggtct tcctggtcca caaggtccaa ttggtcctcc tggtgaaaaa 2460ggaccacaag gaaaaccagg acttgctgga cttcctggtg ctgatgggcc tcctggtcat 2520cctgggaaag aaggccagtc tggagaaaag ggggctctgg gtccccctgg tccacaaggt 2580cctattggat acccgggccc ccggggagta aagggagcag atggtgtcag aggtctcaag 2640ggatctaaag gtgaaaaggg tgaagatggt tttccaggat tcaaaggtga catgggtcta 2700aaaggtgaca gaggagaagt tggtcaaatt ggcccaagag gggaagatgg ccctgaagga 2760cccaaaggtc gagcaggccc aactggagac ccaggtcctt caggtcaagc aggagaaaag 2820ggaaaacttg gagttccagg attaccagga tatccaggaa gacaaggtcc aaagggttcc 2880actggattcc ctgggtttcc aggtgccaat ggagagaaag gtgcacgggg agtagctggc 2940aaaccaggcc ctcggggtca gcgtggtcca acgggtcctc gaggttcaag aggtgcaaga 3000ggtcccactg ggaaacctgg gccaaagggc acttcaggtg gcgatggccc tcctggccct 3060ccaggtgaaa gaggtcctca aggacctcag ggtccagttg gattccctgg accaaaaggc 3120cctcctggac cacctgggaa ggatgggctg ccaggacacc ctgggcaacg tggggagact 3180ggatttcaag gcaagaccgg ccctcctggg ccagggggag tggttggacc acagggacca 3240accggtgaga ctggtccaat aggggaacgt gggcatcctg gccctcctgg ccctcctggt 3300gagcaaggtc ttcctggtgc tgcaggaaaa gaaggtgcaa agggtgatcc aggtcctcaa 3360ggtatctcag ggaaagatgg accagcagga ttacgtggtt tcccagggga aagaggtctt 3420cctggagctc agggtgcacc tggactgaaa ggaggggaag gtccccaggg cccaccaggt 3480ccagttggct caccaggaga acgtgggtca gcaggtacag ctggcccaat tggtttacca 3540gggcgcccgg gacctcaggg tcctcctggt ccagctggag agaaaggtgc tcctggagaa 3600aaaggtcccc aagggcctgc agggagagat ggagttcaag gtcctgttgg tctcccaggg 3660ccagctggtc ctgccggctc ccctggggaa gacggagaca agggtgaaat tggtgagccg 3720ggacaaaaag gcagcaaggg tgacaaggga gaaaatggcc ctcccggtcc cccaggtctt 3780caaggaccag ttggtgcccc tggaattgct ggaggtgatg gtgaaccagg tcctagagga 3840cagcagggga tgtttgggca aaaaggtgat gagggtgcca gaggcttccc tggacctcct 3900ggtccaatag gtcttcaggg tctgccaggc ccacctggtg aaaaaggtga aaatggggat 3960gttggtccca tggggccacc tggtcctcca ggcccaagag gccctcaagg tcccaatgga 4020gctgatggac cacaaggacc cccagggtct gttggttcag ttggtggtgt tggagaaaag 4080ggtgaacctg gagaagcagg gaacccaggg cctcctgggg aagcaggtgt aggcggtccc 4140aaaggagaaa gaggagagaa aggggaagct ggtccacctg gagctgctgg acctccaggt 4200gccaaggggc caccaggtga tgatggccct aagggtaacc cgggtcctgt tggttttcct 4260ggagatcctg gtcctcctgg ggaacctggc cctgcaggtc aagatggtgt tggtggtgac 4320aagggtgaag atggagatcc tggtcaaccg ggtcctcctg gcccatctgg tgaggctggc 4380ccaccaggtc ctcctggaaa acgaggtcct cctggagctg caggtgcaga gggaagacaa 4440ggtgaaaaag gtgctaaggg ggaagcaggt gcagaaggtc ctcctggaaa aaccggccca 4500gtcggtcctc agggacctgc aggaaagcct ggtccagaag gtcttcgggg catccctggt 4560cctgtgggag aacaaggtct ccctggagct gcaggccaag atggaccacc tggtcctatg 4620ggacctcctg gcttacctgg tctcaaaggt gaccctggct ccaagggtga aaagggacat 4680cctggtttaa ttggcctgat tggtcctcca ggagaacaag gggaaaaagg tgaccgaggg 4740ctccctggaa ctcaaggatc tccaggagca aaaggggatg ggggaattcc tggtcctgct 4800ggtcccttag gtccacctgg tcctccaggt ttaccaggtc ctcaaggccc aaagggtaac 4860aaaggctcta ctggacccgc tggccagaaa ggtgacagtg gtcttccagg gcctcctggg 4920tctccaggtc cacctggtga agtcattcag cctttaccaa tcttgtcctc caaaaaaacg 4980agaagacata ctgaaggcat gcaagcagat gcagatgata atattcttga ttactcggat 5040ggaatggaag aaatatttgg ttccctcaat tccctgaaac aagacattga gcatatgaaa 5100tttccaatgg gtactcagac caatccagcc cgaacttgta aagacctgca actcagccat 5160cctgacttcc cagatggtga atattggatt gatcctaacc aaggttgctc aggagattcc 5220ttcaaagttt actgtaattt cacatctggt ggtgagactt gcatttatcc agacaaaaaa 5280tctgagggag taagaatttc atcatggcca aaggagaaac caggaagttg gtttagtgaa 5340tttaagaggg gaaaactgct ttcatactta gatgttgaag gaaattccat caatatggtg 5400caaatgacat tcctgaaact tctgactgcc tctgctcggc aaaatttcac ctaccactgt 5460catcagtcag cagcctggta tgatgtgtca tcaggaagtt atgacaaagc acttcgcttc 5520ctgggatcaa atgatgagga gatgtcctat gacaataatc cttttatcaa aacactgtat 5580gatggttgtg cgtccagaaa aggctatgaa aagactgtca ttgaaatcaa tacaccaaaa 5640attgatcaag tacctattgt tgatgtcatg atcaatgact ttggtgatca gaatcagaag 5700ttcggatttg aagttggtcc tgtttgtttt cttggctaag attaagacaa agaacatatc 5760aaatcaacag aaaatatacc ttggtgccac caacccattt tgtgccacat gcaagttttg 5820aataaggatg gtatagaaaa caacgctgca tatacaggta ccatttagga aataccgatg 5880cctttgtggg ggcagaatca catggcaaaa gctttgaaaa tcataaagat ataagttggt 5940gtggctaaga tggaaacagg gctgattctt gattcccaat tctcaactct ccttttccta 6000tttgaatttc tttggtgctg tagaaaacaa aaaaagaaaa atatatattc ataaaaaata 6060tggtgctcat tctcatccat ccaggatgta ctaaaacagt gtgtttaata aattgtaatt 6120attttgtgta cagttctata ctgttatctg tgtccatttc caaaacttgc acgtgtccct 6180gaattccatc tgactctaat tttatgagaa ttgcagaact ctgatggcaa taaatatatg 6240tattatgaaa aaataaagtt gtaatttctg atgactctaa gtccctttct ttggttaata 6300ataaaatgcc tttgtatata ttgatgttga agagttcaat tatttgatgt cgccaacaaa 6360attctcagag ggcaaaaatc tggaagactt ttggaagcac actctgatca actcttctct 6420gccgacagtc attttgctga atttcagcca aaaatattat gcattttgat gctttattca 6480aggctatacc tcaaactttt tcttctcaga atccaggatt tcacaggata cttgtatata 6540tggaaaacaa gcaagtttat atttttggac agggaaatgt gtgtaagaaa gtatattaac 6600aaatcaatgc ctccgtcaag caaacaatca tatgtatact ttttttctac gttatctcat 6660ctccttgttt tcagtgtgct tcaataatgc aggttaatat taaagatgga aattaagcaa 6720ttatttatga atttgtgcaa tgttagattt tcttatcaat caagttcttg aatttgattc 6780taagttgcat attataacag tctcgaaaat tattttactt gcccaacaaa tattactttt 6840ttcctttcaa gataatttta taaatcattt gacctaccta attgctaaat gaataacata 6900tggtggactg ttattaagag tatttgtttt aagtcattca ggaaaatcta aacttttttt 6960tccactaagg tatttacttt aaggtagctt gaaatagcaa tacaatttaa aaattaaaaa 7020ctgaattttg tatctatttt aagtaatata tgtaagactt gaaaataaat gttttatttc 7080ttatataaag tgttaaatta attgatacca gatttcactg gaacagtttc aactgataat 7140ttatgacaaa agaacatacc tgtaatattg aaattaaaaa gtgaaatttg tcataaagaa 7200tttcttttat ttttgaaatc gagtttgtaa atgtcctttt aagaagggag atatgaatcc 7260aataaataaa ctcaagtctt ggctacctgg a 7291111538DNAHomo sapiens 11ccgtgcaccg tgtgtgcgcg cggcgttgaa atgccctgca cgtcggggca gcgggacaga 60tcccagggtg cccagggagt ctccaagtgc ctcactcctc ccgccgcaaa catgacagag 120aactccgaca aagttcccat tgccctggtg ggacctgatg acgtggaatt ctgcagcccc 180ccggcgtacg ctacgctgac ggtgaagccc tccagccccg cgcggctgct caaggtggga 240gccgtggtcc tcatttcggg agctgtgctg ctgctctttg gggccatcgg ggccttctac 300ttctggaagg ggagcgacag tcacatttac aatgtccatt acaccatgag tatcaatggg 360aaattacaag atgggtcaat ggaaatagac gctgggaaca acttggagac ctttaaaatg 420ggaagtggag ctgaagaagc aattgcagtt aatgatttcc agaatggcat cacaggaatt 480cgttttgctg gaggagagaa gtgctacatt aaagcgcaag tgaaggctcg tattcctgag 540gtgggcgccg tgaccaaaca gagcatctcc tccaaactgg aaggcaagat catgccagtc 600aaatatgaag aaaattctct tatctgggtg gctgtagatc agcctgtgaa ggacaacagc 660ttcttgagtt ctaaggtgtt agaactctgc ggtgaccttc ctattttctg gcttaaacca 720acctatccaa aagaaatcca gagggaaaga agagaagtgg taagaaaaat tgttccaact 780accacaaaaa gaccacacag tggaccacgg agcaacccag gcgctggaag actgaataat 840gaaaccagac ccagtgttca agaggactca caagccttca atcctgataa tccttatcat 900cagcaggaag gggaaagcat gacattcgac cctagactgg atcacgaagg aatctgttgt 960atagaatgta ggcggagcta cacccactgc cagaagatct gtgaacccct ggggggctat 1020tacccatggc cttataatta tcaaggctgc cgttcggcct gcagagtcat catgccatgt 1080agctggtggg tggcccgtat cttgggcatg gtgtgaaatc acttcatata tcatgtgctg 1140taaaataaga actagctgaa gagacaacca aagaagcatt aaggcaggtt gatgctgatg 1200ggaccataaa atatttttac actcaacctg agcggttatt cttgacactc ttaacagaat 1260tttttcaatt gttttccaga actttagtat atgcaaatgt actgaaaggg tagttcaagt 1320ctaaaatgcc ataacccctt tattatttgt tattttttat ttgcattgct ttgccataag 1380tcttcccttg cttgtatctt ccaaagctat tttgaaataa acatgaaaat ttacagtttg 1440ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1538121135DNAHomo sapiens 12gggcagaggg aataagaggc tgcctctgcc caccagtcct gccgcccagg acccgcagca 60gagacgacgc ctgcagcaag gagaccagga aggggtgaga caaggaagag gatgtctgag 120ctggagaagg ccatggtggc cctcatcgac gttttccacc aatattctgg aagggaggga 180gacaagcaca agctgaagaa atccgaactg aaggagctca tcaacaatga gctttcccat 240ttcttagagg aaatcaaaga gcaggaggtt gtggacaaag tcatggaaac actggacaat 300gatggagacg gcgaatgtga cttccaggaa ttcatggcct ttgttgccat ggttactact 360gcctgccacg agttctttga acatgagtga gattagaaag cagccaaacc tttcctgtaa 420cagagacggt catgcaagaa agcagacagc aagggcttgc agcctagtag gagctgagct 480ttccagccgt gttgtagcta attaggaagc ttgatttgct ttgtgattga aaaattgaaa 540acctctttcc aaaggctgtt ttaacggcct gcatcattct ttctgctata ttaggcctgt 600gtgtaagctg actggcccca gggactcttg ttaacagtaa cttaggagtc aggtctcagt 660gataaagcgt gcaccgtgca gcccgccatg gccgtgtaga ccctaacccg gagggaaccc 720tgactacaga aattaccccg gggcaccctt aaaacttcca ctacctttaa aaaacaaagc 780cttatccagc attatttgaa aacactgctg ttctttaaat gcgttcctca tccatgcaga 840taacagctgg ttggccggtg tggccctgca agggcgtggt ggcttcggcc tgcttcccgg 900gatgcgcctg atcaccaggt gaacgctcag cgctggcagc gctcctggaa aaagcaactc 960catcagaact cgcaatccga gccagctctg ggggctccag cgtggcctcc gtgacccatg 1020cgattcaagt cgcggctgca ggatccttgc ctccaacgtg cctccagcac atgcggcttc 1080cgagggcact accgggggct ctgagccacc gcgagggcct gcgttcaata aaaag 1135132889DNAHomo sapiens 13ctggctgccg gctgctgcca ccgcaatccc ggctcctaaa tcagcgcggg gaggcgctcc 60ctccccacgc ccggctctcc gggctctcgg ggccgcgatt ggccgcgccg gcgcccccca 120ccccgggccc ccggctccag ctgccgcgcc attggctgcg ggcctccgcc agcctttaca 180taagaccggg cgcgctcgag tggagttgta taaagcgagc gcgcggcgtc ggggcgggag 240gctcgaggcc agcccgggac cggggctggg agcaagcagg cggcggcgcc ggcggcagag 300gcggcagcga gcgcccgctt cccacgcccc taggcggcgg ggccgagagc gggaggatgg 360ctccgagcgc tgaccccggc atgtccagga tgttaccgtt cctgctgctg ctctggtttc 420tgcccatcac tgaggggtcc cagcgggctg aacccatgtt cactgcagtc accaactcag 480ttctgcctcc tgactatgac agtaatccca cccagctcaa ctatggtgtg gcagttactg 540atgtggacca tgatggggac tttgagatcg tcgtggcggg gtacaatgga cccaacctgg 600ttctgaagta tgaccgggcc cagaagcggc tggtgaacat cgcggtcgat gagcgcagct 660caccctacta cgcgctgcgg gaccggcagg ggaacgccat cggggtcaca gcctgcgaca 720tcgacgggga cggccgggag gagatctact tcctcaacac caataatgcc ttctcggggg 780tggccacgta caccgacaag ttgttcaagt tccgcaataa ccggtgggaa gacatcctga 840gcgatgaggt caacgtggcc cgtggtgtgg ccagcctctt tgccggacgc tctgtggcct 900gtgtggacag aaagggctct ggacgctact ctatctacat tgccaattac gcctacggta 960atgtgggccc tgatgccctc attgaaatgg accctgaggc cagtgacctc tcccggggca 1020ttctggcgct cagagatgtg gctgctgagg ctggggtcag caaatataca gggggccgag 1080gcgtcagcgt gggccccatc ctcagcagca gtgcctcgga tatcttctgc gacaatgaga 1140atgggcctaa cttccttttc cacaaccggg gcgatggcac ctttgtggac gctgcggcca 1200gtgctggtgt ggacgacccc caccagcatg ggcgaggtgt cgccctggct gacttcaacc 1260gtgatggcaa agtggacatc gtctatggca actggaatgg cccccaccgc ctctatctgc 1320aaatgagcac ccatgggaag gtccgcttcc gggacatcgc ctcacccaag ttctccatgc 1380cctcccctgt ccgcacggtc atcaccgccg actttgacaa tgaccaggag ctggagatct 1440tcttcaacaa cattgcctac cgcagctcct cagccaaccg cctcttccgc gtcatccgta 1500gagagcacgg agaccccctc atcgaggagc tcaatcccgg cgacgccttg gagcctgagg 1560gccggggcac agggggtgtg gtgaccgact tcgacggaga cgggatgctg gacctcatct 1620tgtcccatgg agagtccatg gctcagccgc tgtccgtctt ccggggcaat cagggcttca 1680acaacaactg gctgcgagtg gtgccacgca cccggtttgg ggcctttgcc aggggagcta 1740aggtcgtgct ctacaccaag aagagtgggg cccacctgag gatcatcgac gggggctcag 1800gctacctgtg tgagatggag cccgtggcac actttggcct ggggaaggat gaagccagca 1860gtgtggaggt gacgtggcca gatggcaaga tggtgagccg gaacgtggcc agcggggaga 1920tgaactcagt gctggagatc ctctaccccc gggatgagga cacacttcag gacccagccc 1980cactggagtg tggccaagga ttctcccagc aggaaaatgg ccattgcatg gacaccaatg 2040aatgcatcca gttcccattc gtgtgccctc gagacaagcc cgtatgtgtc aacacctatg 2100gaagctacag gtgccggacc aacaagaagt gcagtcgggg ctacgagccc aacgaggatg 2160gcacagcctg cgtggggact ctcggccagt caccgggccc ccgccccacc acccccaccg 2220ctgctgctgc cactgccgct gctgctgccg ctgctggagc tgccactgct gcaccggtcc 2280tcgtagatgg agatctcaat ctggggtcgg tggttaagga gagctgcgag cccagctgct 2340gagcaggggt gggacatgaa ccagcggatg gagtccagca ggggagtggg aaagtgggct 2400tgtgctgctg cctagacagt agggatgtaa aggcctggga gctagaccct ccccaagccc 2460atccatgcac attacttagc taacaattag ggagactcgt aaggccaggc cctgtgctgg 2520gcacatagct gtgatcacag cagacagggt cgctgccctg atggcgctta cattccagtg 2580ggtctaatga ccatatctta ggacacagat gtgcccaggg aggtggtgtc actgcacagg 2640aagtatgagg actttagtgt cctgagttca aatcctgatt caggaactca caaagctatg 2700tgaccttaca ccagtcactt aacttgttag ccatccatta tcgcatctgc aaaatgggga 2760ttaagaatag aatcttgggg ttagtgtgga gattagatta aatgtatgta agacacttgg 2820cacaaaacct ggcacatagt aaaggctcaa taaaaacaag tgcctctcac tgggctttgt 2880caacacgtg 2889143935DNAHomo sapiens 14ggagagccga aagcggagct cgaaactgac tggaaacttc agtggcgcgg agactcgcca 60gtttcaaccc cggaaacttt tctttgcagg aggagaagag aaggggtgca agcgccccca 120cttttgctct ttttcctccc ctcctcctcc tctccaattc gcctcccccc acttggagcg 180ggcagctgtg aactggccac cccgcgcctt cctaagtgct cgccgcggta gccggccgac 240gcgccagctt ccccgggagc cgcttgctcc gcatccgggc agccgagggg agaggagccc 300gcgcctcgag tccccgagcc gccgcggctt ctcgcctttc ccggccacca gccccctgcc 360ccgggcccgc gtatgaatct cctggacccc ttcatgaaga tgaccgacga gcaggagaag 420ggcctgtccg gcgcccccag ccccaccatg tccgaggact ccgcgggctc gccctgcccg 480tcgggctccg gctcggacac cgagaacacg cggccccagg agaacacgtt ccccaagggc 540gagcccgatc tgaagaagga gagcgaggag gacaagttcc ccgtgtgcat ccgcgaggcg 600gtcagccagg tgctcaaagg ctacgactgg acgctggtgc ccatgccggt gcgcgtcaac 660ggctccagca agaacaagcc gcacgtcaag cggcccatga acgccttcat ggtgtgggcg 720caggcggcgc gcaggaagct cgcggaccag tacccgcact tgcacaacgc cgagctcagc 780aagacgctgg gcaagctctg gagacttctg aacgagagcg agaagcggcc cttcgtggag 840gaggcggagc ggctgcgcgt gcagcacaag aaggaccacc cggattacaa gtaccagccg 900cggcggagga agtcggtgaa gaacgggcag gcggaggcag aggaggccac ggagcagacg 960cacatctccc ccaacgccat cttcaaggcg ctgcaggccg actcgccaca ctcctcctcc 1020ggcatgagcg aggtgcactc ccccggcgag cactcggggc aatcccaggg cccaccgacc 1080ccacccacca cccccaaaac cgacgtgcag ccgggcaagg ctgacctgaa gcgagagggg 1140cgccccttgc cagagggggg cagacagccc cctatcgact tccgcgacgt ggacatcggc 1200gagctgagca gcgacgtcat ctccaacatc gagaccttcg atgtcaacga gtttgaccag 1260tacctgccgc ccaacggcca cccgggggtg ccggccacgc acggccaggt cacctacacg 1320ggcagctacg gcatcagcag caccgcggcc accccggcga gcgcgggcca cgtgtggatg 1380tccaagcagc aggcgccgcc gccacccccg cagcagcccc cacaggcccc gccggccccg 1440caggcgcccc cgcagccgca ggcggcgccc ccacagcagc cggcggcacc cccgcagcag 1500ccacaggcgc acacgctgac cacgctgagc agcgagccgg gccagtccca gcgaacgcac 1560atcaagacgg agcagctgag ccccagccac tacagcgagc agcagcagca ctcgccccaa 1620cagatcgcct acagcccctt caacctccca cactacagcc cctcctaccc gcccatcacc 1680cgctcacagt acgactacac cgaccaccag aactccagct cctactacag ccacgcggca 1740ggccagggca ccggcctcta ctccaccttc acctacatga accccgctca gcgccccatg 1800tacaccccca tcgccgacac ctctggggtc ccttccatcc cgcagaccca cagcccccag 1860cactgggaac aacccgtcta cacacagctc actcgacctt gaggaggcct cccacgaagg 1920gcgaagatgg ccgagatgat cctaaaaata accgaagaaa gagaggacca accagaattc 1980cctttggaca tttgtgtttt tttgtttttt tattttgttt tgttttttct tcttcttctt 2040cttccttaaa gacatttaag ctaaaggcaa ctcgtaccca aatttccaag acacaaacat 2100gacctatcca agcgcattac ccacttgtgg ccaatcagtg gccaggccaa ccttggctaa 2160atggagcagc gaaatcaacg agaaactgga ctttttaaac cctcttcaga gcaagcgtgg 2220aggatgatgg agaatcgtgt gatcagtgtg ctaaatctct ctgcctgttt ggactttgta 2280attatttttt tagcagtaat taaagaaaaa agtcctctgt gaggaatatt ctctatttta 2340aatattttta gtatgtactg tgtatgattc attaccattt tgaggggatt tatacatatt 2400tttagataaa attaaatgct cttatttttc caacagctaa actactctta gttgaacagt 2460gtgccctagc ttttcttgca accagagtat ttttgtacag atttgctttc tcttacaaaa 2520agaaaaaaaa aatcctgttg tattaacatt taaaaacaga attgtgttat gtgatcagtt 2580ttgggggtta actttgctta attcctcagg ctttgcgatt taaggaggag ctgccttaaa 2640aaaaaataaa ggccttattt tgcaattatg ggagtaaaca atagtctaga gaagcatttg 2700gtaagcttta tcatatatat attttttaaa gaagagaaaa acaccttgag ccttaaaacg 2760gtgctgctgg gaaacatttg cactctttta gtgcatttcc tcctgccttt gcttgttcac 2820tgcagtctta agaaagaggt aaaaggcaag caaaggagat gaaatctgtt ctgggaatgt 2880ttcagcagcc aataagtgcc cgagcacact gcccccggtt gcctgcctgg gccccatgtg 2940gaaggcagat gcctgctcgc tctgtcacct gtgcctctca gaacaccagc agttaacctt 3000caagacattc cacttgctaa aattatttat tttgtaagga gaggttttaa ttaaaacaaa 3060aaaaaattct tttttttttt tttttccaat tttaccttct ttaaaatagg ttgttggagc 3120tttcctcaaa gggtatggtc atctgttgtt aaattatgtt cttaactgta accagttttt 3180ttttatttat ctctttaatc tttttttatt attaaaagca agtttctttg tattcctcac 3240cctagatttg tataaatgcc tttttgtcca tccctttttt ctttgttgtt tttgttgaaa 3300acaaactgga aacttgtttc tttttttgta taaatgagag attgcaaatg tagtgtatca 3360ctgagtcatt tgcagtgttt tctgccacag acctttgggc tgccttatat tgtgtgtgtg 3420tgtgggtgtg tgtgtgtttt gacacaaaaa caatgcaagc atgtgtcatc catatttctc 3480tacatcttct cttggagtga gggaggctac ctggagggga tcagcccact gacagacctt 3540aatcttaatt actgctgtgg ctagagagtt tgaggattgc tttttaaaaa agacagcaaa 3600cttttttttt tatttaaaaa aagatatatt aacagtttta gaagtcagta gaataaaatc 3660ttaaagcact cataatatgg

catccttcaa tttctgtata aaagcagatc tttttaaaaa 3720gatacttctg taacttaaga aacctggcat ttaaatcata ttttgtcttt aggtaaaagc 3780tttggtttgt gttcgtgttt tgtttgtttc acttgtttcc ctcccagccc caaacctttt 3840gttctctccg tgaaacttac ctttcccttt ttctttctct tttttttttt tgtatattat 3900tgtttacaat aaatatacat tgcattaaaa agaaa 3935158034DNAHomo sapiens 15cttgagtgtt agagctgagt agttttccca gaatctctaa gtccttttta tgctctttta 60tgaatgaata gaattagtaa aagataaata aattttttct tttggatttc ttaaccagtg 120gaaaaaatgt tgactttaaa agttcataaa atcaaatttt gcttaagaat atgttatttc 180cacttgtgag gccagcctgg tagacctctg ggatcctttt ctgttcactc acacaccact 240gagataagga gtgaagtgtg ggctaaatag ggctgaggct tgggcaaggg catttctgcc 300agagcaccag agacgtcagc atctcaaggg cactgtggta tggaaaagga cgccacatga 360gtaaatttta aaaatataaa tattttaaag ggtaaaaatg agggtccctg tatttgagga 420tataaaagat gaaactgaag aagaaaagat aggggaagaa gaaaatgaag aagaccaggt 480cttctataag cctgttattg aagacttaag catggaattg gccagaaaat gcacggaact 540cattagcgat atccgttata aagaagagtt taaaaagtcc aaggataagt gtacatttgt 600gactgacagt cctatgctaa accatgtaaa aaatatcggt gcttttattt ctgaggcaaa 660atacaaaggc accattaaag ctgacctttc taattctctt tataagcgga tgccagccac 720aattgacagt gtttttgcag gagaagttac acagctccag agtgaggtgg cctacaagca 780gaaacatgat gctgccaaag gattctcaga ttatgcccac atgaaggagc cccctgaggt 840taaacatgcc atggaggtca ataaacacca gagtaatatt tcttatagga aagacgtgca 900ggacacccac acgtacagtg cagaacttga ccgaccagac atcaagatgg caacccagat 960ctctaagatc ataagcaatg cagaatacaa gaaaggacaa ggaataatga ataaagagcc 1020cgctgtaatt ggaagaccag attttgaaca tgccgtggaa gcttctaaac tttctagtca 1080aattaaatac aaagaaaaat tcgataatga aatgaaggat aagaaacatc attacaatcc 1140tcttgaaagt gcttctttta ggcagaatca gcttgctgct acactggcga gcaatgtgaa 1200gtacaagaaa gacattcaaa atatgcatga tccagtttca gatctcccaa atttgttgtt 1260tttagaccat gttttgaaag ccagcaaaat gctcagcggc cgagaatata aaaagctctt 1320tgaggaaaac aaaggaatgt atcattttga tgcagatgct gtggaacatc tgcaccataa 1380aggcaatgcc gtcctccaaa gtcaggtgaa atataaagaa gaatatgaga aaaataaggg 1440aaagccaatg cttgaatttg ttgagacacc atcatatcaa gcttcaaagg aggctcaaaa 1500gatgcaaagt gaaaaagttt acaaagagga ttttgagaag gagattaaag gaaggtcatc 1560actggattta gacaagactc cagaattttt acatgtaaag tacatcacca accttctgag 1620ggagaaagaa tataaaaaag atttggaaaa tgagataaaa gggaaaggaa tggaacttaa 1680ttcagaagtt cttgatatcc aaagagcaaa gcgggcctct gaaatggcaa gtgagaaaga 1740atacaagaaa gacctggagt caataattaa agggaaagga atgcaagctg gcactgacac 1800ccttgaaatg cagcatgcca agaaggctgc agagatagcg agtgagaaag actataaaag 1860agatctggag actgaaatta aagggaaagg gatgcaggtg agcacagaca ctcttgatgt 1920ccagagagct aagaaagcat ccgagatggc cagccagaaa caatacaaga aggacttaga 1980aaatgaaatt aaagggaaag gaatgcaagt gagcatggat atcccagata tccttcgagc 2040caagaggaca tctgaaatct atagccagag aaagtataaa gatgaagcag agaagatgct 2100ttctaactat tctaccatag cagatactcc tgaaattcag agaattaaga caactcaaca 2160aaacattagt gcggtatttt ataagaaaga agtgggagct ggcactgcag tgaaagatag 2220cccagagatc gaacgagtga agaaaaatca gcagaatatt agttcagtga aatacaaaga 2280agagattaaa catgcaacag ccatttctga tcctccagaa ctaaagagag ttaaagaaaa 2340ccagaagaac atcagcaatc tccagtataa agagcaaaac tacaaggcca ctccggtaag 2400catgaccccg gagatagaga gagtgaggcg aaaccaggag cagctgagtg cggtaaaata 2460taagggagaa cttcaacggg gaactgcaat ttctgatcca ccagagctga agagggcaaa 2520agaaaaccag aaaaacatca gcaatgttta ttacagaggt cagctgggaa gagctaccac 2580tttaagtgta actcctgaaa tggaaagagt gaagaagaat caagaaaata ttagctcggt 2640aaaatatacc caggaccata aacagatgaa aggtagacca agtctgattt tagatacacc 2700tgctatgaga catgttaaag aagcacaaaa tcatatttca atggtaaaat accatgaaga 2760ttttgaaaaa acaaagggga gaggctttac tcccgtcgtg gacgatcctg tgacagagag 2820agtgaggaag aacacccagg tggtcagcga tgctgcctat aaaggggtcc accctcacat 2880cgtggagatg gacaggagac ctggaatcat tgttgacctc aaagtttggc gcacagatcc 2940tggctccatc ttcgaccttg atcccctgga agacaatatt cagtctagaa gtctccatat 3000gctctctgaa aaggcgagtc actataggcg acactggtct cgatcccatt ccagcagtac 3060tttcggtaca ggtctcggag acgacaggtc agaaatctcc gagatttacc ctagcttttc 3120atgctgcagt gaggtaacaa gaccgtctga tgaaggagca cctgttcttc ccggagccta 3180tcagcaaagc cattcccaag gctatggcta catgcaccag accagtgtgt catccatgag 3240atcaatgcag cattcaccaa atctaaggac ctaccgagcc atgtacgatt acagtgccca 3300ggatgaagac gaggtctcct ttagagacgg cgactacatc gtcaacgtgc agcctattga 3360cgatggctgg atgtacggca cagtgcagag aacagggaga acaggaatgc tcccagcgaa 3420ttacattgag tttgttaatt aattatttct ccctgccctt tgagctttat tctaatgtat 3480cccaaaccta atctttttaa aagatagaag atacttttaa gacaacttgg ccattatttt 3540acaatgatgt atccttcctt tgacaattag acacacaggt accaggaaga aggaatgacc 3600tctgggctga aaacagcagc attttcagta attcctacaa acaaaaatct ttgtgtctgg 3660acgcctggtg ctgctaattg tgttcatggt ttcctttgat tggctattga acccttctgg 3720gaaatgtatt tttgtagact ttaatagaga agttgattgt cccttaaatg tagcgtgtgt 3780ttgaaacttc ttagctgtca ctttggaatc accccaagcc aattctctta actctgtaat 3840gcagccaata atacaaaccc gttttgcttt tgagtcatga ggcaatttcc aatattagtg 3900aaaattgccc aatataataa gtgtaaacag tggcagaagg acagtctggt taaaattata 3960ttgactggtg gccttaggga tctagaaact tctactaaac agagaaattt ccttgttccc 4020taggctgact ggtatctatt tatttctcat ttgtaccaag gcatctccta ctctccattt 4080atattctatg gacccaagtc tatgctcagt tccacagaat gtcaggacca aataacttca 4140cagctactct gcaaagggca aattataatg tcattgatat aatttcccta gtagcattta 4200ccctgttgca tgtcatgtag attcaagctt ctgtaacata ggcagctgca ctgcgcgttc 4260ctattattga agcaaaaagg gtgactgata cctaaaagcc ctttcttcct ctagtcgcca 4320gctcatcaga aaaacatact ttgaaaagat gcttgagatt ttcctgctgc atcgcactct 4380agtttggaag gatttacatc ttaggaaata acatgtatac tctagtaaat aagcgattta 4440ggtgttccat tgaacagctt tgattaactt aatgccacca ttgatttcaa agtgaagaaa 4500atgtaacaga agccagtgaa gcaatggaag ctggagtgtg actggaaaaa tactcagcaa 4560acaaagttac caattccata cagagatgat ctggtgtctt cttttggaaa atggtattca 4620aattctggaa tggaaatcta gccaccaaaa cgggttaatc aaaagacgtc cttttccgtt 4680ttttttgctt ttattttcta aatcattttt aagggaatga aacaggaatg tcatcagaga 4740ttttttagta caggcccaag agcctgtact ctaagaaaga aatttttgcc atgtatgaat 4800tttcgaataa gtgactttgc aggcttttgc tagcccttgc tggtgggtct ggaaattaca 4860tccagagtct gcagtccagg tcaccaagcc agcggcaccc gtcggcaacc ctgtgtttaa 4920cggattgtgc cgtttactgt gacctgcaac ggggtggcat tcacttaggg tctgacttca 4980cagctatgac aaaaccgaaa aagcaaaact gcgaggaagt gctaagatgt acgggtcttg 5040gggatatctg ccttatatgt tatattcaag gaaattaacg aaacatcctg tgaaacatcg 5100tttaaggaaa cgtttactag tccaaaggcc aaagctaatt tatttccact ttagaaaagt 5160tagcacatgc ttttgaaaat ctgtgatttc attttattag gctaaaaggg taaataggct 5220ttattacact gaagctgcat ctatatgtca ctgacataaa gttgaaaaaa taaatgcagg 5280caaataacta gagacttctt ttaagggggt ttggctggtt tctctcactg aaatggccag 5340tcgtgattaa agtgataaaa ccccatatct gttttggtat attgtacaca aacctacaaa 5400aataaactga acttgcaata tttttgcaat aaaatctgtc gttaaaactg aggataaaat 5460acctgctcaa ttttatttta ctaagtatat atttacattt cacccaggca ggccattttc 5520ttttgtgatt ataagaaaga gtagttgttg attaaatttt cagactaaat ataggacagg 5580tacaattttg gataaatagc agatttataa gaaccgcaat gaaaactgac ttgaaataat 5640gcttgtaatc aggaaagtaa tttcatccac cgatttcaaa accagattca ctgagcataa 5700aagtcaatac atatttgagg aataagtctc ctaaaatttt aagcttcacg taataatgtt 5760tgcatagcaa aatatttctg cttcaagcct ttaggaatta agatctgatc agaatttaac 5820taaagggtag ttgttttaca atgaagacta aaactgaaca agatgttgca tgcgcttagg 5880ccataatttg gtagtgttgg cagttgttaa taaagcttgt caggatgtta agcatctcag 5940gagaaatatt ggaaaattat atgtataaaa ccaaagtgct gtttttaaaa gcatcattta 6000aaaaaaaatg acatgcctga acaacttttc cactttccac gtgcttccct cccacctttg 6060gtttggcaac aggtatctcg tgcatgaagc tgacagctaa agaagatttt aaaaattgag 6120ttaaagatga ctgtgtaaat gtccaagcac agagagcatg cacctgactt tctaaagttt 6180gatgtgttct caagcctgac agaagcacaa ggaacagttt gatacacttt taaaaggttc 6240tgaaaacaaa gctgtatagg gatcctctct ctcttgagca aagtatagca acagaatata 6300ttgcttttgt tgtaagcttt tgtagtacat gtttttacta ataattcttg ttctctagaa 6360agctttctat ttctaaccta tggcaaaatg aatccttcat gtcttcttgt tattgtttac 6420acacttgcag tgtagcccag tttgaaatat ttatttggtt atcaactgcc catggaggag 6480gctcttgatg atcccaggtc tcctcgacct ccatacacca cacaggcatt tgtaagcaca 6540gtttccacaa gcaccttgta ggaatatgga taagattaga ccagcccctc tctgtccact 6600gggtttattt cttgaagaag atgcagatct ggtttttcca atgtgccaca gtctttcctt 6660atcctctcca tgctgagctt gacaacactc tgggaatgag gaacaagact ttttctaaaa 6720agatagtgga agttcaaggg atgtacctcg ttttcaggtt catccatctc cagtggaatg 6780ttttcaataa aagatgaaga aaatgtgtgt gatctttaat aacacatccc tatagaaagt 6840ggataaaaga tataccaaaa ctgtaataca gatatataca aatataggtg cctttttgat 6900tactcttgtt tgtctagtat gctcttggaa agaaaaccaa gcaagcaagt tgctgcctat 6960tctatagtaa tattttatta cacatgattg atatttttgt ggtagggaag tgggatgctc 7020ctcagatatt aaaggtgtta gctgattgta ttttatctct aaagatttag aactttagaa 7080aatgccgact tcttccatct atttctgaaa ggttctttgt ggatttatat agagttgagc 7140tatataaaca ttaactttag atttgggatt taaaatgcct attgtaagat agaataattg 7200tgaggctgga ttcactacac aagatgaact tcacttcata aattaattat accttagcga 7260tttgcttctg ataatctaaa agtggctaga ttgtggttgt tttggttaag gtgatatgga 7320ggtgggagag cttttagtta agtaagaagc tatgtaaact gacaaggatg ctaaaataaa 7380agtctctgaa gtattccatg ccttttggac cctttcctcg caactaactg tcaactgttg 7440atcaaaaaag tcaaggcatt gtatgttgct tctgtggtta ttattctgtg atgcttagac 7500tacttgaacc cataaacttg gaagaatctt tgagcaaatt ttctcagttg tctgtatgac 7560ttcagtatat tcctgggaat gccataggat tttttgtgct tgatacatgg tatccagttt 7620gcatagtatc acttctttgt aatccagttg ctgttaagaa tgatgtactt taaaggaaaa 7680gagaaaactg catcacagtc ccattctcca gtgtccatgc aatgaattgc tgagcattta 7740ggaagcagca ccaagtctat tacaggcatg gtgtgaaact tgatgtttga cctgtgatca 7800aaattgaacc attgtacagt ttggcttctg tttgcttcaa aatatgtaga attgtggttg 7860atgattaatt tgcgagacta actttgagag tgtaacagtt ttgaagaaaa cattgaatgt 7920tttacaaatg aaggggcttc acggaatgtt acaatgttac taatataatt tggcttttgt 7980tatgcaaatt gttaacacca gctattaaaa tatattttag tagaaaaaaa aaaa 80341628DNAArtificialHAPLN1 forward primer 16tgaaggatta gaagatgata ctgttgtg 281720DNAArtificialHAPLN1 reverse primer 17gccccagtcg tggaaagtaa 201818DNAArtificialHAPLN1 probe sequence 18tacaaggtgt ggtattcc 181922DNAArtificialMFAP5 forward primer sequence 19cgaggagacg atgtgactca ag 222020DNAArtificialMFAP5 reverse primer sequence 20agcgggatca ttcaccagat 202117DNAArtificialMFAP5 probe sequence 21acattcacag aagatcc 172220DNAartificialPrimer sequence 22gccaaggtgt tttcacacag 202340DNAArtificialPrimer sequence 23taatacgact cactataggg gccaaggtgt tttcacacag 402420DNAArtificialPrimer sequence 24ctctgaagca gtagacacca 202521DNAArtificialPrimer sequence 25cctagcctgg ctttcttgct c 212641DNAArtificialPrimer sequence 26taatacgact cactataggg cctagcctgg ctttcttgct c 412721DNAArtificialPrimer sequence 27ccattgggtc tctgcaaatc c 21

Claims

1. A method of evaluating the composition of a cell culture, said method comprising: a) obtaining a plurality of cells from a cell culture; b) determining the average expression level of a fibroblast marker in the plurality of cells, said fibroblast marker being such that its normalized expression levels are lower in chondrocytes than in fibroblasts or synoviocytes; and c) determining the composition of the culture based on the average expression level of the fibroblast marker, wherein: 1) the cell culture comprises chondrocytes or 2) the fibroblast marker is microfibrillar associated protein 5 (MFAP5).

2.-35. (canceled)

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