Identification Of The Mutant Gene Vangl2^m1Yzcm And The Research Of Homozygous Mutants’ Death

ENU (ethylnitrosourea) is a kind of alkylating agent,causing gene mutation effectively.In some labs researchers use ENU to obtain various gene mutant mice.After screening we could obtain abundant G1mice with different phenotype,which are used to investigate gene function and set up animal model of human disease.In our lab,we injected ENU in mice intraperitoneally and generated a mutant mice with loop-tail phenotype.To find out the relationship between gene mutation and the loop-tail phenotype,we did the following research.As a result,we found out that loop-tail phenotype was caused by the mutation of Vangl2gene.In the further research,we confirmed that the death of homozygotes is due to the obviously opened neural tube of Vang12mlYzcm/Vangl2mIYzcm homozygotes.The study includes the following two parts:1.Mapping and identification of mutation gene for loop-tail phenotypeWbct mutant was isolated as a phenotype inherited in a dominant fashion in an experimental screen.Mating Wbct heterozygotes with Wildtype B6,we gained a new mutant with loop-tail and white spotting phenotype.Interestingly,after breeding this new mutants with Wildtype B6,loop-tail mice without white spotting phenotype was obtained.To confirm the mode of inheritance (recessive or dominant),the loop-tail mice was crossed with Wildtype B6,C3He/J(C3H) mice.We generated103mice by breeding heterozygotes with B6.Although50%transmission of the dominant mutation is expected,only32offsprings showed loop-tail phenotype.These datas clarified that the mutation of LP mice was inherited as semidominant trait,with only31.1%transmission in B6genetic background.The No.of loop-tail mice were obviously lessened or disappeared in F1(B6LP/+×C3H) mutants.We only obtained one F1(B6LP/+×C3H) mutants.These results suggest that the phenotype is related to the genetic background and that the mutation is only partially penetrant on B6genetic background.In addition,this penetrance is much lower in C3H mice.Based on the position of pax3in chr1,DNA pools from10[(B6×C3H)F1×B6] N2,(B6×C3H)F2heterozygotes respectively were screened with two microsatellite markers(D1Mit113and DlMitl49).Our experiments resulted in no recombination using D1Mit113and DlMit149primers.Thus,the mutation was localized in the79.54cM region between D1Mit113and D1Mitl49.To find out the related genes with loop-tail phenotype,we searched the mouse gene informatics (MGI) database.Considering the result of gene location screened by the two microsatelite markers,we confirmed that the mutation of loop-tail mice located in chr1.Indeed, the phenotype of vangl2mutant mice in the79.54cM region was similar to that of loop-tail mice,which indicates that vangl2is a very strong candidate gene for the observed phenotype.To search for the mutation in the candidate genes, exons of vangl2from LP heterozygotes were synthesized by PCR amplification.PCR products were subjected to2%agarose gel electrophoresis,excised from the agarose gel, purified using QIAgen kit and were directly sequenced.The resulting DNA sequences were analyzed with DNAstar program,by comparing with the sequence of Wildtype B6.The result showed that the mutant line had a a single C to T base substitution, converting codon449from CAG to UAG,a nonsense mutation,compared with the published sequence, as well as to normal B6.2. Study of Vang12mlYzcm/Vang12mlYzcmhomozygotes’deathThe point mutation(C→T) in vangl2yielded an FspBI (BfaI) restriction site. DNA extracted from the embryos was amplified and the PCR products were digested by FspBI enzyme.Digestion of the PCR product with FspBI was expected to generate DNA fragments as the following sizes:wildtype without FspBI (BfaI) restriction site only had one band of452bp;heterozygotes with bands of452bp,133bp,319bp;homozygotes with two bands of133bp,319bp.In our study we used FspBI (BfaI) enzyme to cut452bp DNA strap extracted from the embryos yielded by intercrossing LP heterozygous mice.As a result,we succeeded in identifying three different genotypes.In the homozygotes death research,we found that none alive homozygous mice were obtained.Therefore,we confirmed that the homozygous mutation of Vangl2mlYzcm gene caused the death of Vang12mlYzcm/Vangl2mlYzcm homozygotes.It is reported that the homozygous mutation of Vang12gene may be lethal perinatally in mice with a phenotype of completely open neural tube from midbrain to tail.To confirm the suspect,we intercrossed LP heterozygous mice and laparotomized E12.5d and E18.5d mice.The result confirmed that Vang12mIYzcm/Vangl2mlYzcm mice were lethal perinatally, and most of which died in uterus E18.5d.E18.5d Vang12mIYzcm Vangl2mlYzcm embryos revealed a malformed neural tube persistently opening from hindbrain to tail,which is clearly distinguishable from the closed neural tube in the wildtype littermates.The smaller overall size of the homozygous embryo suggests overall growth retardation.Histological examination of transverse sections through embryos at E12.5illustrates an enlarged floor plate, an exposed neuroepithelium, and the obviously opened neural tube.