Functional compatibility of Rho family genes in vivo.

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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.