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

一种基因陷阱方法来识别参与中枢神经系统发育的新基因

基本信息

批准号：
06454698
负责人：
KUWANO Ryozo
金额：
$ 4.22万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454698/
关键词：
gene gene trap nervous system development ES cells neomycin resistant chimeric mice embryonic leathal immunohistochemistry 神経初期発 G418

项目摘要

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.

采用基因陷阱法筛选出一个与中枢神经系统发育有关的新基因。以小鼠E-钙粘蛋白基因、新霉素核糖基转移酶（neo）、β-半乳糖苷酶（lacZ）和脑心肌炎病毒IRES基因为剪接受体，构建诱捕载体。采用电穿孔法将基因捕获载体导入小鼠胚胎干细胞。用G418筛选ES细胞，并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、MAP 2和胶质细胞酸性蛋白）的抗体进行免疫化学检测。在13个G418抗性细胞系中，有3个细胞系（GT 3 - 8、11和12）呈免疫反应阳性，并同时显示lacZ基因的表达。其中lacZ在GT 3 - 8、11和12的中枢神经系统中检测到表达。GT3-11和12，而不是GT3-8，被传递到生殖系。用质粒拯救法从ES细胞中分离出被诱捕的基因，用5 'RACE法获得与基因诱捕载体融合的cDNA，并通过DNA测序和数据库检索，在GT 3 -11和GT 12中诱捕到新基因。全长cDNA和捕获基因的克隆及其结构分析正在进行中。