Breeding, Phenotyping And Identifying The Mutant Gene Of Venter-yellow Pigmentation Mouse

The venter-yellow pigmentation mouse(VYslac)was a new mutant found during normal breeding of the C57BL/6J (B6) mice and isolated by the staff of Shanghai Laboratory Animal Center, Chinese Academy of Science, which showed single gene autosomal dominant genetic pattern, yellow coat on the ventral surface of head, neck and trunk,while the rest part was black and consistent with the background strain. Preliminary work of our laboratory has mapped the mutant gene in the region of5256015bp between the149804749th bp and the155060764th bp from the centromere in chromosome2. We bred homozygous mice on the basis of early experiment, initially compared the phenotypic differences among homozygotes, heterozygotes and wild type mice, as well as expanded sample size for mapping, screened higher density genetic markers to narrow down the mutant gene to a smaller region, and identified three of candidate genes. What we did would help to establish a novel model for human disease and lay a good foundation for gene function research. The work is summarized as follows:1. Breeding and phenotyping of homozygous mice(VYslac-m/m)Heterozygous (GO) intercrossed, selected20who have venter-yellow phenotype (heterozygous or homozygous) randomly in the offspring(G1), mated with background strain B6, if G2mice appeared normal phenotype, G1was heterozygous; if G2(more than20) all appeared venter-yellow phenotype, G1was homozygous. We totally filtered out five homozygous mice, two of which were male, three were female. Homozygous (G1) mated, then the same litter mated successive, we had bred homozygous mice to7th generation. On this basis, compared the phenotype of homozygous, heterozygous and B6, pigmentation and structure of back hair between homozygous and B6were not significantly different, pigmentation of venter hair was lighter than B6, while structure was normal. There were not significantly different between homozygous and B6on the number of melanocytes and melanin content in the hair follicles of the back skin, while less than B6in the venter skin, distributed sparse.We didn’t find abnormal in position, morphology,structure and histopathology between homozygous and B6.There was no obvious differences between VYslac-m/m and VYslac-m-,suggesting the mutant was dominance inherited completely.2. Fine mapping and preliminary identifying the candidate gene of VYslac micePreliminary work of our laboratory has mapped the mutant gene in the region of5256015bp between the149804749th bp and155060764th bp from the centromere in chromosome2, Mated VYslac with DBA/2to get F1generation, then backcrossed to DBA/2J to expanded the sample size of F2generation for mapping to526, selected SNP markers and microsatellite markers between D2mit307and D2mit310, the region we had mapped before.D2mit285, D2mit495, D2mit411,and rs27310903were usable. After linkage analysising, mutant gene was mapped to the region between154711297bp and155235028bp from the centromere. Through querying the mouse genome database we found only9genes within the region:Gm14217, Gm14214, Raly, Eif2s2, Agouti, Ahcy, Rik, Itch and Gm14227. Mice with autosomal dominant mutation of Agouti gene accompanied with varying degrees of yellow hair due to overexpression of Agouti signaling protein ASP.Furthermore, Raly and Eif2s2can also affect the expression of Agouti gene and generate yellow spot. Agouti, Raly and Eif2s2were confirmed as candidate genes, Through designing primers, applying PCR amplification and sequencing the mRNA and genomic DNA, but we found no mutation in the coding region. The results suggest that there is another proposed allele gene between154711297bp and155235028bp from the centromere, or there is important and regulatory sequence in non-coding region, relevant work will be further carried out.