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.

在这项研究中，我们建立了一个有条件的基因靶向系统，用于人类结节硬化症2（TSC2）基因的小鼠同源物。最初，通过基因靶向技术生成了带有TSC2基因的静音突变等位基因（S-Allele）的小鼠线，其中外显子3和4侧面是一对LoxP序列。由于CRE介导的重组从S替代产生的缺乏外显子3和4的缺失突变等位基因（D-贵族）在功能上被灭活，因为D-异雷氏杂合的小鼠在先前报道的TSC2基因敲除小鼠中杂合形成了肾肿瘤。 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表现出异常呼吸后，到1岁。对照小鼠均未显示这种死亡率。在CKO小鼠的心脏中，通过CRE介导的重组产生了D-优质。 CKO小鼠的心脏明显扩大，并显示出组织学异常，例如肥大和心肌细胞的核异常。此外，CKO小鼠的心脏通过超声心动图显示了左心室的扩张，并表达的失败心脏基因，例如心房努力疗多肽（ANP）基因。因此，类似于扩张的心肌病的表型是由小鼠心肌特异性TSC2敲除诱导的。现在，我们进一步分析了TSC2产物在心肌细胞生长和分化中的功能。本研究中建立的TSC2的条件基因将是一个有用的实验系统，旨在阐明TSC2产物的功能以及与TSC2突变相关的肿瘤发生机理。