Functional analysis of neuronal migration-associated genes in murine brains by in utero gene transfer techniques with GFP reporter genes.

啮齿类动物出生前暴露于低剂量电离辐射可导致皮质组织发生过程中神经元迁移速度减慢，并导致成熟大脑皮质结构紊乱。我们试图通过两种分子生物学技术，即限制性地标基因扫描(RLCS)方法和抑制消减PCR方法，来阐明X射线照射后胎鼠脑内表达模式发生变化的基因。我们已经扫描了大约13,000个基因，发现在扫描的基因中，有20多个基因在X射线照射的胚胎和未照射的胚胎之间存在差异表达。显示上调的基因之一是Rab6A，它被认为与跨高尔基网络的囊泡运输有关。此外，RabKIFL和GAPCenA基因表达上调。然后我们建立了一种基因转染方法，在这种方法中，绿色荧光蛋白标记的蛋白可以在培养细胞中表达，从而产生显性的负面效应。基于这些结果，我们应用了一种宫内电穿孔技术，将带有EGFP标记的感兴趣基因引入小鼠胚胎大脑，这项研究目前正在进行中。

Prenatal exposure to ionizing radiation of low doses in rodents induces deceleration of neuronal migration during cortical histogenesis, and results in disorganized cortical architecture in mature brain. We have attempted to elucidate genes which show changing patterns of expression after exposure to X-rays in fetal mouse brains, by applying two molecular biological techniques, i.e., the restriction landmark cDNA scanning (RLCS) method and the suppression subtractive PCR method.Approximately 13,000 cDNA species have been scanned and it turned out that more than twenty genes among the genes scanned are differentially expressed between X-irradiated embryos and non-irradiated ones. One of the genes showing up-regulation is Rab6A which is known to be associated with vesicle transport from trans-Golgi network. In addition, RabKIFL and GAPCenA genes were up-regulated. We then established a gene transfection method in which EGFP-tagged proteins of interest can be expressed in cultured cells to produce dominant negative effects. Based on these results we have applied an in utero electroporation technique to introduce genes of interest tagged with EGFP to embryonic mouse brains and this study is currently under way.