|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1999 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1998 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1997 : ¥1,300,000 (Direct Cost : ¥1,300,000)
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.