| Research Abstract |
We constructed three long SAGE (Serial Analysis of Gene Expression) libraries from undifferentiated ATDC5 cells at day 2, mature ATDC5 cells at day 21, and hypertrophic/calcified ATDC5 cells at day 42. A total of 143,095 SAGE tags (71,646 and 71,449 tags from undifferentiated (day 2) and mature (day 21) libraries, respectively) were analyzed and 1868 transcripts were differentially represented in the two libraries (p<0.05). In an analysis of a total of 135,544 SAGE tags (67,660 and 67,884 tags from mature (day 21) and hypertrophic/calcified (day 42) libraries, respectively), 1070 tags were identified as differentially expressed transcripts in the two libraries (p<0.05). These differentially expressed transcripts include genes for extracellular matrices, transcription factors, cyotoskeletons, intracellular molecules, secreted factors, and receptors. The roles of most genes in chondrogenic differentiation are previously unidentified. We performed in situ hybridization on the sections of mouse embryos at E16.5 and confirmed the expression of abundantly expressed genes such as extracellular matrices in cartilage. For the validation of genes (transcription factors, secreted factors, and receptors) expressed at relatively low levels, we examined the expression of these genes in ATDC5 cells (undifferentiation, mature, and hypertrophic/calcified), primary chondrocytes, and rib cartilage by northern blot analysis. For the functional analysis of the genes identified by SAGE analysis, we established the ATDC5 cell transient expression system using electroporation. Taking advantage of in ovo electroporation in the chick limb bud, we succeeded in the establishment of the assay system to evaluate cartilage formation and interactions between the cartilaginous primodium and surrounding tissues including blood vessels.
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