Study on ECM tenascin-family proteins by gene knockout

点击翻译按钮获取中文摘要

The tenascins are a growing family of extracellular matrix glycoproteins.To date three members of the family have been identified in mammals : tenascin-C (TN-C) (also called cytotactin), tenascin-R (TN-R) (for restrictin), and tenascin-X (TN-X).We have identified that TN-X gene is localized in the class III region of the major histocompatibility complex locus and the expression of TN-X is often reciprocal to that of TN-C in the developing mouse embryo.In order to test the possible involvement of TN-X in embryonic migrations and morphogenetic processes and in later events such as wound healing and oncogenesis, we have used homologous recombination in embryonic stem (ES) cells to inactivate the TN-X gene.Now we are attempting to generate strains of mice deficient in TN-X.Thus far we have constructed the targeting vectors which disrupt TN-X gene by deletion of the 5' portion removing the translation initiation codon.By using of the targeting vector, we have identified the two targeted clones among 1200 G418-resistant transfectants analyzed.To generate mice harboring the targeted TN-X allele, these targeted ES cell clones were injected into host 8-cell stage embryo.However no extensive ES-cell contribution was observed with the two clones as assayd by coat color chimerism, indicating that all chimeric embryos with high ES-cell contribution die between E13 and E16.The reason why these chimeric embryos die earlier in gestation remains to be determined.Further experiments should help to clarify this issue.As for the regulation of TN-X expression, Dr.Chiquet-Ehrismann (Friedrich Miescher Institute, Switzerland) has investigated the expression of TN-X in fibroblasts and carcinoma cells in culture.None of the growth factors or cytokines examined affected the expression level of TN-X,however, steroid hormone glucocorticoids, were found to downregulate TN-X mRNA levels and protein synthesis in fibroblasts but not carcinoma cells at physiological concentrations.