Genetic Studies On The Spontaneous White Spotting Mutantion Of B6Strain In Mouse

Mouse coat coloration variants including albino，black，agouti，yellow，and piebeld,etc. It has been studied for over a century. These mice have proven to bevaluable assets to geneticists and embryologists and have been instrumental for manyimportant discoveries. White spotting mutant mouse has the same phenotype andmechanism with some Human pigmentation disorders （piebaldism, Waardenburgsyndrome WS）, so it serves as an ideal disease model of human diseases.The spontaneous white spotting mutant mice of our laboratory belong to B6strain background. Mating test experiment confirmed that the mutant phenotype wasstable and could be inherited. We determined the heredity mode of the white spottingphenotype, screened and observed for homozygous mutant mice. By mappingmutation gene, we confirmed the candidate gene （kit）. And then we cloned andsequenced the kit cDNA, finally found the mutant nucleotide position. Our researchnot only successfully identified the molecular mechanism of this mutation, but alsoprovided a new material for kit gene function and related human diseases’ research.This thesis consists of several following parts.1、Determining the heredity mode of this white spotting mutant phenotypeThe heterozygous white spotting mutant mice were mated with normal B6strainmice.146offspring of G1generation mice were born. By counting the numbers ofmutant phenotype and normal phenotype mice and comparing with the theoreticalvalue of single dominant gene heredity mode for Chi-square test, we finallydetermined single dominant gene heredity mode of this mutant phenotype.2、Screening and observing of the homozygous mutant miceWe used heterozygous mutant mice intercrossed for reproducing homozygousmice and finally got72offspring in16litter sizes. Among the offsprings, newphenotype or expanded white spotting mice were not observed. Mating testexperiment indicated that there were no homozygous mutant offsprings and itsuggested that homozygous mutants were dead owing to gene purity. We found someabnormal offspring of1²-day-old by observating and speculated they werehomozygous mutant mice which might die of anemia. 3、Mapping and cloning the white spotting mutant geneThe mutant mice mated with PWK strain mice reproducing F1generationmutants. N₂generation mice were gained by backcrossing F1generation mutants withPWK mice. All tail genome DNA of N₂mice were amplified by primers of the38microsatellites. We evaluated the relationship between the mutant phenotype and themicrosatellites （linkage or recombination） by calculating log odds score （LODS）, itwas to found that the mutant gene was mapped on mouse chromosome5.Subsequently, with the number of N₂being up to216, the microsatellites D5Mit304and D5Mit359which were closely next to centromere42cM of chromosome5wereselected and linkage analysed with mutant gene. Finally, we infered that the mutantgene lied between D5Mit304and D5Mit359,42.20cM from the centromere. Byanalysising these genes′function, we confirmed the candidate kit gene.Then the kitgene cDNA was cloned and sequenced, the results showed that there was a G to Atransition at cDNA nucleotide position2576, causing a G to R change at amino acid822.