Identification Of Wbct White Spots Mice Mutation Gene And Study Of Homozygous Mutants' Embryonic Death

Pax3 is an important regulatory factor in migration,generation,differentiation of myogenic precursor cells and development of neuroderm in embryos.The mutation of Pax3 in human and mice can result in neural crest dysplasia during embryomic period causing a variety of abnormal phenotypes.It's closely related to some diseases,such as Warrdenburg Syndrom,rhabdomyosar and neural tube defects.Wbct mouse is a kind of white spot mutation mouse which was obtained by ENU mutagenesis.In the previous study,Wu Baojin et al located the mutation gene on mouse chromosome 1,about 41.6cM from the centromere.Based on their study,we identified the heredity mode of Wbct white spot phenotype and determined that the phenotypes were caused by the mutation gene.Based on the successful identification of mutation gene,we also established the genotyping method for Wbct mutants,Wbct homozygous embryonic deaths were observed and analyzed.This thesis consists of two parts.1.Accurate positioning and identification of mutation gene for Wbct white spots phenotypeThe heterozygous mice(B6 background) were mated with normal B6 mice.The numbers of mutants and normal mice of G1 generation were counted and were compared with the theoretical value of single dominant gene heredity mode for Chi-square test in order to determine the genetic model.The F1 generation mutants with tails albino were gained by mating Wbct heterozygous mice(B6 background) with normal D2 mice,then N2 generation mutants were gained by mating F1 generation mutants with normal B6 mice.The DNA of N2 generation mice was extracted.We chose microsatellites D1Mit180(41cM from centromere)and D1Mit415(52cM from centromere) as well as PCR to amplify the genome DNA of the N2 generation mutantse for accurate positioning.Nine exon sequences of Wbct heterozygous mice Pax3 gene were amplified by PCR and sequenced after gel recovery.We used DNA star software to analyse the Pax3 gene sequences of Wbct mice and predict the change of amino acids due to die bases altering.The results showed in B6 background,the major phenotypes of Wbct heterozygous mice were tails albino and large white belly spots.In D2 background,(B6×D2)F1 generation mutants showed tails albino,but white spots were obviously decreased or diapeared; [(B6×D2)F1×B6]N2 generation mice showed tails albino, and sizes of white belly spots had significant differences which indicated that the involvement of D2 background had some compensation for the development disabilities of melanocytes.Microsatellites tests showed the recombinant frequency of D1Mit180 and mutation gene wa(s3.19±2.51)%,the recombinant frequency of D1Mit415 and mutation gene wa(s9.04±4.10)%.There were totally 23 cases of recombination and the frequency wa(s12.23±4.68)%.The mutation gene was located at 43.87cM from the centromere.By gene function analysis,we found that phenotypes caused by mutation of Pax3 gene,which was located at 44cM,were very similar to the phenotypes of Wbct.So the Pax3 gene was identified as strong candidate gene for Wbct.PCR products were sequenced and results showed that there was a clear overlap on the Pax3 exon2 sequence diagram of heterozygous mice.Using DNA star software to analyse the sequencing results,it showed that on one chromosome of Wbct heterozygous,the 88th codon on the Pax3 gene coding area changed from UGC to termination codon UGA which leaded to early termination of protein coding and this was the reason for Wbct white spot phenotype.2.Preliminary study of Wbct homozygous mutants'embryonic deathWe used heterozygous mutants backcrossed with B6 for reproducing heterozygous mice and heterozygous mutants intercrossed for reproducing homozygous mice;the embryo morphologic observation was performed by selecting 10.5d and 12.5 embryos;the DNA of Wbct intercross offsprings was extracted.As Wbct heterozygous mice Pax3 gene exon2 changed from C to A,the HpyCH4V enzyme cutting site was disappeared.We used the site as a target to design primer and amplify fragment.After enzyme cutting PCR products by HpyCH4V incision enzyme,the results were compared with the theoretical value of single dominant gene heredity mode for Chi-square test in order to determine whether it fitted the law of free combination.The results showed there were 127 offsprings by intercrossing Wbct heterozygous mutant mice,and no new phenotype or expanded white spots were observed.55 offspings were mated with normal B6 mice and normal color offprings were seen.It indicated that there were no homozygous mutation offspings and it suggested that homozygous mutants were dead during embryonic period.In offspings obtained by intercrossing Wbct with B6 mice,we detected a kind of abnormal mice which showed bending tails phenotype.This abnormal mice's tails bended towards their back and this phenotype hadn't been reported yet.In the abnormal embryos,the blood vessels of amnion were clearly reduced and the color of amnion was very pale,the sizes were obviously smaller than normal embryos;some of the abnormal embryos had head introcession,this is a typical performance of NTDs.We calculated the results of enzyme cutting tests,and by Chi-square test we determined that the genetic separation ratio was consistent with the free combination law of 1∶2∶1.In summary,Wbct mice provided important research materials for further study of Pax3 gene function and Warrdenburg syndrome,NTDs and was very valuable for establishing animal models of human diseases.