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识别小鼠胚胎中新的发育调控基因的有力手段。捕获的克隆可以分析标记基因在体内和体外的表达模式。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的表达模式不同。因此，有必要利用靶基因的DNA片段作为探针来确认其表达模式。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陷阱克隆分析的原位RT-PCR方法的开发，但在这个项目中我们可以发现一些新的证据。(1)为了阐明消化道的前后（AP）模式，我们系统地检查了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共线性。Hox基因在后期（12.5 d.p.c）的表达边界与个体肠道亚域的形态边界相对应。(2)利用3种诱捕媒介共分离诱捕克隆109个。这些克隆体中的一个；Ayu8008在ES细胞中表现出较强的表达，在小鼠胚胎中表现出8.5、9.5、15.5的组织特异性表达模式。捕获基因与大鼠EST （A1548797）序列具有高度同源性。(3) Ayu8008纯合子突变小鼠出现生长迟缓。这个小鼠系的表型似乎受到遗传背景的影响。

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.