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

Establishment of novel conditional gene targeting methods using Cre-loxP system

利用Cre-loxP系统建立新型条件基因打靶方法

基本信息

批准号：
07458216
负责人：
NODA Tetsuo
金额：
$ 4.48万
依托单位：
Tohoku University (1997)Japanese Foundation For Cancer Research (1995-1996)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458216/
关键词：
gene targeting Cre recombinase loxP sequences recombinant adenovirus transgenic mouse keratin 14 Purkinje cell specific protein 2 olfactory marker protein Cre遺伝子ジーンターゲティングケラチン14遺伝子 PO蛋白遺伝子アデノウイルスベクターコンディショナル・ノックアウト L7遺伝子

项目摘要

An establishment of a gene targeting technology allows us to introduce mutations in any target genes on mouse chromosomal DNA.However, since many genes function in developmental stages of mice, mutant mice often suffer embryonic lethality and, therefore, it is difficult to analyze functions of these genes in adult tissues. Conditional gene targeting was invented to circumvent this problem. In this study, we established novel technologies to achieve tissue or cell-lineage specific gene targeting in mice. Our system is based on DNA recombination mediated by Cre recombinase and its recognition sequences, loxP.A pair of loxP sites was introduced into target genes on mouse chromosomal DNA using conventional gene targeting technology. A transgenic allele, which may confer LacZ expression upon Gre-loxP mediated recombination, was also used to validate established technology in this study. To express Cre gene specifically in particular tissues or cells in mice, we developed two novel systems. First, we constructed a recombinant adenovirus carrying Ore gene driven by strong and ubiquitous promoter. By the infection of this virus, we could specifically introduce mutations in target genes in various organs of mice, such as liver, lung, skin, pancreas and intestinal tract. Although the efficiency of gene inactivation is not so high (0.1-3%), we could precisely regulate an onset of gene inactivation using this method. As an altemative way, we also established several lines of transgenic mice, each of which may express Ore recombinase in specific lineage of mice. In this system, promoters of keratin 14 (K14), Purkinje cell specific protein 2 (PCP2), and olfactory marker protein (OMP) genes were successfully used to express Ore gene in epidermal basal cells, cerebellar Purkinje cells and olfactory nerve cells, respectively.

基因打靶技术的建立使我们能够在小鼠染色体DNA上的任何靶基因中引入突变。然而，由于许多基因在小鼠的发育阶段起作用，突变小鼠经常遭受胚胎致死，因此，很难分析这些基因在成年组织中的功能。有条件的基因靶向技术就是为了解决这个问题而发明的。在这项研究中，我们建立了新的技术，以实现组织或细胞系特异性基因靶向小鼠。该系统基于Cre重组酶及其识别序列loxP介导的DNA重组，利用常规基因打靶技术将一对loxP位点引入小鼠染色体DNA上的目的基因。一个转基因等位基因，它可以赋予LacZ表达后Gre-loxP介导的重组，也被用来验证本研究中建立的技术。为了在小鼠特定组织或细胞中特异性表达Cre基因，我们开发了两种新的系统。首先，我们构建了一个携带Ore基因的重组腺病毒，该基因由强启动子驱动。通过该病毒的感染，我们可以特异性地在小鼠的各个器官，如肝、肺、皮肤、胰腺和肠道中引入靶基因突变。虽然基因失活的效率不是很高（0.1-3%），但我们可以使用这种方法精确地调节基因失活的开始。作为一种替代方法，我们还建立了几个品系的转基因小鼠，每个品系都可以在特定的小鼠谱系中表达Ore重组酶。在该系统中，角蛋白14（K14），浦肯野细胞特异性蛋白2（PCP 2）和嗅觉标记蛋白（OMP）基因的启动子被成功地用于表达Ore基因在表皮基底细胞，小脑浦肯野细胞和嗅神经细胞分别。