A gene trap approach to identify a new gene involved in central nervous system development

利用基因陷阱的方法鉴定了一个参与中枢神经系统发育的新基因。以小鼠e -钙粘蛋白基因剪接受体、新霉素核糖基转移酶（neo）、β -半胱氨酸苷酶（lacZ）和脑心肌炎病毒基因衍生的IRES为载体，构建了诱捕载体。采用电穿孔法将基因诱捕载体导入小鼠胚胎干细胞。用G418筛选ES细胞，使其在悬浮培养系统中分化，并用抗脑特异性蛋白（巢蛋白、神经丝蛋白、MAP2和胶质纤维酸性蛋白）抗体进行免疫组织化学检测。13株G418耐药细胞株中有3株（GT3-8、11和12）免疫反应，同时出现lacZ基因表达。将13种ES细胞系注入野生型C57BL/6囊胚中生成嵌合小鼠，并在14.5 dpc嵌合小鼠胚胎中分析了诱捕基因的表达谱。其中，在GT3-8、11、12的中枢神经系统检测到lacZ的表达。GT3-11和gt3 - 12，而不是GT3-8，可以进入种系。用质粒拯救法从胚胎干细胞中分离捕获基因，用5′race法获得与基因捕获载体融合的cDNA。从GT3-11和gt3 - 12的部分DNA序列和数据库检索中发现了新的基因。全长cdna和捕获基因的克隆及其结构分析正在进行中。

A gene trap approach is used to identify a new gene involved in development of central nervous system. A trap vector was constructed with a splicing acceptor of mouse E-cadherin gene, neomycin ribosyl transferase (neo), beta-galacosidase (lacZ) and IRES derived from encephalomyocarditis virus gene. The gene trap vector were introduced into mouse embryonic stem (ES) cells by electroporation. ES cells were selected with G418 and allowed to differentiate in suspension culture system and immunohistochemically examined with antibodies against brain specific proteins (nestine, neurofilament, MAP2 and glial fibrillary acidic protein). Of 13 G418 resistant cell lines 3 cell lines (GT3-8,11 and 12) were immunoreactive and simultaneously manifested lacZ gene expression.Chimeric mice were generated by injection of the 13 ES cell lines into wild type C57BL/6 blastulas and an expression pattern of the trap gene was analyzed at 14.5 dpc embryos of chimeric mice. Among them lacZ expression was detected in the central nervous system of GT3-8,11, and 12. The GT3-11 and 12, but not GT3-8, were transmitted into the germ line. The trapped genes were isolated from ES cells by plasmid rescue and the cDNA fused with the gene trap vector were obtained by 5'RACE.Novel gene were trapped in both GT3-11 and 12 from these partial DNA sequences and data base search. Cloning of the full length cDNAs and the trapped gene and their structural analyzes are now in progress.