权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Functional analysis of Tsc2 gene product by conditional gene targeting.

通过条件基因打靶对 Tsc2 基因产物进行功能分析。

基本信息

批准号：
10680783
负责人：
KOBAYASHI Toshiyuki
金额：
$ 1.92万
依托单位：
The Japanese Foundation for Cancer Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

In this study, we established a conditional gene targeting system for the mouse homologue of human tuberous sclerosis 2 (Tsc2) gene. Initially, a mouse line carrying a silent mutant allele (S-allele) of Tsc2 gene, in which exons 3 and 4 were flanked with a pair of loxP sequences, was generated through gene targeting technique. A deletion mutant allele (D-allele) lacking exons 3 and 4, generated from the S-allele by Cre-mediated recombination, was functionally inactivated, since mice heterozygous for D-allele developed renal tumors as Tsc2 knockout mice previously reported. Next, we crossed mice carrying S-allele with "knock-in" mice which express Cre recombinase under the promoter of ventricular-specific myosin light chain gene (Mlc2v) to carry out the cardiac muscle-specific Tsc2 knockout in vivo.Although mice homozygous for S-allele carrying Mlc2v-Cre allele (CKO mice) were born and grown normally, they were dead by 1 year of age after exhibiting abnormal breathing. None of the control mice showed such mortality. In hearts of CKO mice, D-allele was generated by Cre-mediated recombination. The hearts of CKO mice were markedly enlarged and showed histological anomalies such as hypertrophy and nuclear atypia of cardiac muscle cells. In addition, the hearts of CKO mice showed dilation of left ventricle by echocardiography and expressed maker genes of failing heart such as atrial natriuretic polypeptide (Anp) gene. Thus, phenotype resembling the dilated cardiomyopathy was induced by the cardiac muscle-specific Tsc2 knockout in mice. Now we further analyzed the function of Tsc2 product in growth and differentiation of cardiac muscle cells. The conditional gene targeting of Tsc2 established in this study will be a useful experimental system to elucidate the function of Tsc2 product as well as the mechanism of tumorigenesis associated with Tsc2 mutation.

本研究建立了人结节性硬化症2（Tsc 2）基因小鼠同源基因的条件性基因打靶系统。首先，通过基因打靶技术产生携带Tsc 2基因的沉默突变等位基因（S等位基因）的小鼠品系，其中外显子3和4侧接有一对loxP序列。缺失突变等位基因（D-等位基因）缺乏外显子3和4，从S-等位基因通过Cre介导的重组产生的，功能失活，因为小鼠杂合的D-等位基因发展肾肿瘤Tsc 2敲除小鼠先前报道。接着，将携带S等位基因的小鼠与在心室特异性肌球蛋白轻链基因（Mlc 2 v）的启动子下表达Cre重组酶的“敲入”小鼠杂交，进行心肌特异性Tsc 2的体内敲除。携带Mlc 2 v-Cre等位基因的S等位基因纯合子小鼠（CKO小鼠）虽然出生和生长正常，但在1岁时因出现呼吸异常而死亡。对照组小鼠均未显示出这种死亡率。在CKO小鼠心脏中，通过Cre介导的重组产生D等位基因。CKO小鼠的心脏明显增大，并出现心肌细胞肥大和核小体等组织学异常。此外，CKO小鼠心脏超声心动图显示左心室扩张，并表达心力衰竭标志基因，如心房钠尿多肽（Anp）基因。因此，在小鼠中通过心肌特异性Tsc 2敲除诱导类似扩张型心肌病的表型。本研究进一步分析了Tsc 2产物在心肌细胞生长和分化中的作用。本研究建立的Tsc 2条件性基因打靶技术为进一步研究Tsc 2基因产物的功能以及Tsc 2突变相关的肿瘤发生机制提供了一个有效的实验体系。