Study on ECM tenascin-family proteins by gene knockout

Tenascins是一个细胞外基质糖蛋白家族，目前已在哺乳动物中鉴定出三个成员：Tenascin-C(TN-C)(也称为细胞肌动蛋白)、Tn-R(Tn-R)(表示限制蛋白)、TN-X基因定位于主要组织相容性复合体基因的III类区域，在发育中的小鼠胚胎中，TN-X的表达往往与TN-C的表达相反。为了检测TN-X在胚胎迁移和形态发生过程以及在创伤愈合和肿瘤发生等后续事件中的可能参与，我们利用胚胎干细胞中的同源重组来灭活TN-X基因。现在我们正试图产生TN-X基因缺陷的小鼠品系。到目前为止，我们已经构建了靶向载体，通过删除5‘部分去除翻译起始密码子来干扰TN-X基因。利用靶向载体，我们在分析的1200个G418抗性转基因细胞中鉴定了两个靶向克隆。为了产生目标TN-X等位基因的小鼠，将这些靶向ES细胞克隆注射到宿主8细胞期胚胎中，但通过毛色嵌合体检测，这两个克隆没有观察到广泛的ES细胞贡献对于TN-X表达的调节，瑞士弗里德里希·米歇尔研究所的Chiquet-Ehrismann博士研究了TN-X在成纤维细胞和癌细胞中的表达。所研究的生长因子或细胞因子都没有影响TN-X的表达水平，然而类固醇激素糖皮质激素在生理浓度下下调成纤维细胞中TN-X的mRNA水平和蛋白质合成，但不能下调癌细胞中TN-X的表达。

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.