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Development of the in situ RT-PCR method to aim at the analysis of gene trap clones

开发用于分析基因陷阱克隆的原位 RT-PCR 方法

基本信息

批准号：
09680728
负责人：
ARAKI Masatake
金额：
$ 1.6万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680728/
关键词：
in situ hybridization RT-PCR mouse embryo Hox genes ES cell gene trap Hox遺伝子

项目摘要

Gene trapping in embryonic stem (ES) cells is a powerful means of identifying novel, developmentally regulated genes in mouse embryos, using a promoter-less reporter gene. Trapped clones can be analyzed for expression pattern of the tagged gene in vitro and in vivo. However, in the case of loss of enhancer element of targeted gene, the expression pattern of reporter gene is different from that of targeted gene. Therefore, it is necessary to confirm the expression pattern using a DNA fragment of targeted gene as a probe. In order to detect the mRNA of targeted gene, in situ RT-PCR method were explored for mouse embryos. Though we have not finished yet to develop the in situ RT-PCR method to aim at the analysis of gene trap clones, we could find several new evidence during this project.(1) To elucidate the anteroposterior (AP) patterning of the digestive tract, we have systematically examined the expression patterns of Hox a-7, a-9, b-6, b-7, b-8, b-9, c-6, c-8, c-9, d-8, and d-9. The expression patterns of these genes showed co-linearity along the wall of the digestive tract. The expression boundaries of the Hox genes at later stages (12.5 d.p.c.) corresponded to the morphological boundaries of individual gut subdomains.(2) Using three types of trap vector, we have isolated 109 trap clones. One of these clones; Ayu8008 showed strong expression at ES cell, and tissue specific expression pattern at 8.5 d.p.c., 9.5 d.p.c., and 15.5 d.p.c. mouse embryo. Trapped gene shows high homology with a rat EST (A1548797) sequence.(3) Ayu8008 homozygote mutant mouse showed growth retardation. And the fenotype of this mouse line seems to be effected by genetic background.

胚胎干细胞的基因捕获技术是利用无启动子报告基因在小鼠胚胎中鉴定新的发育调控基因的有力手段。可以在体外和体内分析捕获的克隆的标记基因的表达模式。然而，在靶基因的增强子元件缺失的情况下，报告基因的表达模式与靶基因的表达模式不同。因此，有必要使用靶基因的DNA片段作为探针来确认表达模式。为了检测目的基因的mRNA，探索了小鼠胚胎原位RT-PCR方法。虽然我们还没有完成针对基因诱捕克隆分析的原位RT-PCR方法的开发，但我们可以在这个项目中发现一些新的证据。(1)为了阐明消化道的前后（AP）模式，我们系统地检测了Hox a-7、a-9、B-6、B-7、B-8、B-9、c-6、c-8、c-9、d-8和d-9的表达模式。这些基因的表达模式沿消化道壁呈沿着共线性。Hox基因在后期（12.5 d.p.c.）对应于单个肠亚域的形态学边界。(2)利用三种诱捕载体，共分离到109个诱捕克隆。其中一个克隆Ayu 8008在ES细胞中显示出强表达，并且在8.5 d.p.c.时显示出组织特异性表达模式，9.5 d.p.c.，和15.5 d.p.c.小鼠胚胎捕获的基因与大鼠EST（A1548797）序列高度同源。(3)Ayu 8008纯合子突变小鼠表现为生长迟缓。该品系小鼠的种型受遗传背景的影响。