ENU-induced Mutagenesis In Mouse, And Mapping And Identifying A Novel Kitl^-2Bao Allele Causing White-spotting Phenotype

Ethylnitrosourea(ENU)-induced mutagenesis is an important stratage as phenotype-driven method to study gene functions. A medium scale experiment of ENU-induced mutagenesis in mouse was carried out in our laboratory. Total9kinds of mutant strains were obtained in this project. Kitl-2Bao as one of these mutant genes causing white-spotting was mapped and identified by the ways of genomic scaning and positional candidate cloning respectively. The work is summarized as follows:1.123of C57BL/6J adult male mice (generation0, GO) were treated by intraperitoneal injection of ENU solution. These treated mice were mated with the normal females of C57BL/6J to breed total3048generation1(Gl)mice, and154of the mice showing different mutation phenotypes were selected by careful examination. All survial G1mutants were mated with normal B6, and their offspring were used to evaluate whether they had the parents’abnormal phenotype or not.9kinds of mutants were obtained at last and there were5cases showing white spot,1case showing tail curl and3cases showing abnormal eye. As one of the white spotting mice, the504th mutant mouse presented white spot abdomen, the tail end albino and extremities albinism. This mutant was named as Kitl-2Bao showing single gene autosomal dominant genetic pattern with penetrance being of100%.2. Focusing on Kitl-2Bao as the study material, we used method of linkage analysis to map the mutant gene and found that the LODS was7.08between the D10Mit70and the gene, so linkage was confirmed between them. Mutation gene was further narrowed down to the region between47.06cM and54.72cM from the centromere in chromosome10. According to the phenotype characteristic and the mouse genomic information, Kitl was believed to be the primary candidate gene of white spot. And then, design of primers, RT-PCR amplification of the Kitl mRNA, and sequencing PCR product revealed that the697th base of the Kill ORF converted from G to T, resulting in the corresponding amino acid G could not be synthesized.The mutant mice bred in this experiment are hopefully developed as the models of different human diseases in the future. The results about Kitl-2Bao not only have confirmed its model value as human piebaldism, and enriched the genetic map of mouse, but also provided the solid foundation for the functional study of Kitl.