Acquisition Of A Kind Of Mice With Polydactyly By ENU And Identification Of The Mutant Gene

N-ethyl-N-nitrosourea (ENU) is a kind of synthetic alkylating agents that can efficiently induce random or single base mutation in many species. Lots of mutant mice can be obtained in shorter time by ENU mutagenesis. A kind of polydactylous mutant mice which showed autosomal dominant inheritance has been screened by ENU mutagenesis,the mutant mice present preaxial polydactyly that varied in phenotypic severity.The position of the mutant gene was located on the chromosome, on the basic of Alx4has preliminarily been regarded as the candidate gene of the polydactyly phenotype, sequence analysis of Alx4was performed to determine the Alx4gene mutation was the cardinal reason of polydactyly phenotype. Our work consists of two parts:1. Acquisition of the polydactylous mice and location of the mutant geneA mouse with polydactyly was discovered after screening G1mice by ENU mutagenesis.There were15mice with polydactyly in the71progenies of the polydactylous heterozygote mating with C57BL/6J (B6) wild type.Some mice existed polydactyly phenotype after the G2normal mice mating with B6wild type further.The results have indicated that this mutation was a dominant inheritance of incomplete penetrance.There were4mice showed polydactyly phenotype in the19progenies of the polydactylous B6heterozygote mating with DBA/2J (D2) wild type. And61mice showed polydactyly phenotype in317N2mice,which were obtained from backcrossing F1(B6×D2) with B6.7wild type mice,6homozygotes and12heterozygotes with polydactyly were got in the25progenies of N2polydactylous mice intercrossing.The mutant heterozygotes show an extra digit on the anterior aspect of one hinddigit than normal mice,and severity varies; Homozygotes are more serious in not only the polydactyly(up to7digits)of foredigit and hinddigit but also malformation of hindlimb,in addition,homozygotes were found eyelids opened at birth.Alcian blue and alizarin red staining shows that heterozygotes have an extra digit on the anterior aspect of hindlimb,and homozygotes have polydactyly in four limbs and anomalies of the tibia. Some preaxial digits paralleled with the normal digit and some appeared as a bifurcated first digit. No obvious anomalies were found in other skeletons and cartilages compared with wild type.In order to locate the position of the mutant gene on the chromosome, tail DNA of the[(B6×D2) F1×B6] N2mice were extracted and44microsatellite markers were used to scan the whole genome of the N2polydactylous mice including autosome and sex chromosome X.5of28samples exchanged between the mutant gene and D2mit58in the first linkage analysis,LOD was2.71and was judged to exist linkage possibility,others had no tendency of linkage. On the basic of initial scansion, high density microsatellite markers near the D2mit58were used and the number of N2was enlarged, the further determination showed that5of60samples exchanged between the mutant gene and D2mit184(53.07cM),LOD is10.59;4of60samples exchanged between the mutant gene and D2mit45(50.63cM),LOD is11.68.Finally, the mutant gene was located between D2Mit45and D2Mit184, and Alx4gene was regarded as the candidate gene of polydactyly through local area gene function analysis.2. Sequence analysis of Alx4gene4pairs of primers were designed to aim for the4exons of Alx4gene, genome DNA of Alx4was amplified by PCR,then products were recycled and sequenced.It was found that an obvious A-T overlap signal located at the single point of the heterozygote sequence finally.The sequencing results of RT-PCR were in keep with the sequencing results of genome DNA.The mutation located on the position of433nucleotide in genetic coding areas of Alx4(No.2exon of Alx4),and resulted in the145th codon AAA(encoding lysine)changed to the terminator codon TAA,so it caused premature termination of protein encoding and was determined to the cardinal reason of polydactyly phenotype.