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

胚胎干细胞中的基因捕获是利用无启动子的报告基因在小鼠胚胎中识别新的、受发育调节的基因的有效手段。捕获的克隆可以分析标记基因在体外和体内的表达模式。然而，在靶基因增强子元件缺失的情况下，报告基因的表达模式与靶基因不同。因此，有必要利用目的基因的DNA片段作为探针来确定其表达模式。为了检测目的基因的mRNA，对小鼠胚胎进行了原位RT-PCR方法的探索。虽然我们还没有建立针对基因陷阱克隆的原位RT-PCR方法，但我们在这个项目中发现了一些新的证据：(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管壁呈共线性。HOX基因在后期(12.5D.P.C.)的表达边界与单个肠道亚区的形态边界相对应。(2)利用三种类型的诱捕载体，共分离到109个诱捕克隆。其中Ayu8008在ES细胞中有较强的表达，并且在8.5、9.5和15.5D.P.C.有组织特异性表达。小鼠胚胎。捕获的基因与大鼠EST(A1548797)序列高度同源。(3)Ayu8008纯合子突变小鼠表现出生长迟缓。这一小鼠品系的基因分型似乎受遗传背景的影响。

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.