Functional compatibility of Rho family genes in vivo.

我们开发了可互换的基因捕获系统。报告基因β-geo可以通过Cre-突变的lox定点重组被替换到任何其他感兴趣的基因中。Rho家族蛋白是与RAS相关的小GTP酶，调节多种细胞过程。本研究旨在阐明Rho家族基因在活体内的功能兼容性。杂合子β-geo(Rhoa^&lt；+/geo&gt；)小鼠表现出严重的生长迟缓，大部分在出生后几周内死亡。第一步，将小鼠RhoA的同源物之一酵母的Rho1基因敲入陷阱等位基因而不是β-geo基因。杂合子Rho1敲击小鼠未表现出生长迟缓。在第二步中，人骨骼肌肌动蛋白(α)基因启动子被敲入TRAP等位基因，而不是β-GEO基因。第三步，用TRAP等位基因取代了β-geo基因，插入了小鼠RhoC基因。这些替换可以挽救杂合子的表型。最后，我们测试了报告基因从β-Geo到EGFP的替换，并观察了替换后表型的变化。在杂合子EGFP(Rhoa^&lt；+/BGFP&gt；)小鼠中，该表型完全消失。由于β-Geo和EGFP基因都有较强且普遍存在的表达，β-Geo蛋白的强烈表达可能会严重影响其表型。说明这是RhoA蛋白缺乏症的表型。现在，我们正在研究RHOC替换是否可能挽救纯合子的表型。

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.