Functional compatibility of Rho family genes in vivo.

我们开发了可交换基因陷阱系统。报告基因β-geo可以通过Cre-mutant lox位点特异性重组被替换成任何其他感兴趣的DNA。Ayu17-52小鼠系是通过交换基因诱捕法获得的Rhoa基因敲除小鼠系。Rho家族蛋白是ras相关的小gtpase，调节多种细胞过程。其在体内的功能尚不清楚。本研究项目的目的是明确Rho家族基因在体内的功能相容性。杂合子β-geo（Rhoa^<+/geo>）小鼠表现出严重的生长迟缓，多数在出生后数周内死亡。首先，将小鼠Rhoa的同源基因之一——酵母Rho1基因敲入诱捕等位基因中，而不是敲入β-geo基因。杂合子Rho1敲入小鼠无生长迟缓。第二步，在陷阱等位基因中敲除人骨骼肌α肌动蛋白（HSA）基因启动子，而不是β-geo基因。在第三步，敲除小鼠Rhoc cDNA中的陷阱等位基因，而不是β-geo基因。这些替换可以挽救杂合子的表型。最后，我们测试了将报告基因从β-geo替换为EGFP。更换后，也观察到表型的变化。杂合子EGFP（Rhoa^<+/BGFP>）小鼠的表型完全消失。由于β-geo和EGFP基因的表达都非常强烈且普遍存在，因此β-geo蛋白的强烈表达可能会严重影响表型。纯合子EGFP（Rhoa^<EGFP/EGFP>）小鼠表现出与纯合子Rho1敲入（Rhoa^<Rho1/Rho1>）小鼠相同的胚胎致死性。这意味着这是Rhoa蛋白缺乏的表型。现在，我们正在研究Rhoc替换是否可以挽救纯合子的表型。

We have developed the exchangeable gene trap system. The reporter gene, β-geo, can be replaced into any other DNA of interest through Cre-mutant lox site-specific recombination.The Ayu17-52 mouse line is the Rhoa gene knockout mouse line produced by the exchangeable gene trap method. Rho family proteins are Ras-related small GTPases that regulate a variety of cellular processes. Its function in vivo is still unclear.The purpose of this research project is to clear up the functional compatibility of Rho family genes in vivo.Heterozygous β-geo(Rhoa^<+/geo>) mice showed severe growth retardation, and most of them died in a few weeks after birth.At the first step, Rho1 gene of yeast, one of homologues of mouse Rhoa, was knocked in the trap allele instead of the β-geo gene. Heterozygous Rho1 knock in mice showed no growth retardation. At the second step, human skeletal muscle α actin(HSA) gene promoter was knocked in the trap allele instead of the β-geo gene. At the third step, mouse Rhoc cDNA was knocked in the trap allele instead of the β-geo gene. These replacements could rescue the phenotype of heterozygote. Finaly, we have tested a replacement of reporter gene from β-geo to EGFP.After replacement, the change of the phenotype was observed, too. The phenotype was disappeared completely in heterozygous EGFP(Rhoa^<+/BGFP>) mice. Since the both of β-geo and EGFP gene are expressed quite strongly and ubiquitously, the strong expression of β-geo protein might affect the phenotype severely.Homozygous EGFP(Rhoa^<EGFP/EGFP>) mice show embryonic lethality like as homozygous Rho1 knock in (Rhoa^<Rho1/Rho1>) mice. It means that this is the phenotype of Rhoa protein deficiency. Now, we are investigating if Rhoc replacement might rescue the phenotype of homozygote.