Association Of Goat (Capra Hircus) CD4 Gene Exon 6 Polymorphism With Ability Of Sperm Internalizing Exogenous DNA

The spontaneous capability of sperm cells binding exogenous DNA molecules and internalizing them into nuclei can be exploited by using spermatozoa as vectors for delivering foreign genetic information to eggs during fertilization.Sperm-mediated gene transfer(SMGT) appears to be simple,efficient,and relatively inexpensive methods in modifying animals and the genome of animals,on the other hand it's applicable to every species using spermatozoa for its own propagation.But at the same time there is a great deal of randomness and uncertainty.DNA uptake by the mammalian spermatozoa is a very specific and high regulated phenomenon.Only a small amount of binding DNA Can be internalized into nucleus.It greatly reduces the efficiency of SMGT to produce transgenic animals.The selection of sperm donor also plays an important role in producing transgenic goat.It was found that different goat sperm donors appeared different ability in binding foreign gene.CD4 molecule is one of the transport systems on the sperm plasma membrane.In this study,the standard sperm parameters and the ability in binding foreign gene of bucks were detected one by one,the polymorphism of caprine(Capra hircus) CD4 gene was analyzed and then the correlation between them was evaluated.(1) Seminal samples of 17 individuals from three breed bucks were collected with the pre-warmed artificial vagina.The ejaculates were immediately diluted in pre-warmed TALP buffer and washed at room temperature.The sperm were incubated in vitro with foreign DIG-labeled linear pEGFP-N₁ plasmid under the condition of 1.00μg end-labeled linear DNA plasmid and 10⁶ spermatozoa incubated for 60 min at room temperature.The positive goat spermatozoa were detected by the in situ hybridization methods.(2) The single nucleotide polymorphisms(SNP) sites of exon 6 of caprine CD4 gene were detected by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) methods.(3) The association which of the polymorphism of goat CD4 gene exon 6 with ability of sperm internalizing exogenous DNA was analyzed by Statistical Product and Service Solutions(SPSS) 13.0 for windows.The main results are as following:1.The capability of sperm internalizing exogenous DNA molecules was significantly difference among the three goat breeds.The spermatozoa from Nanjiang Yellow goats had the highest ability in internalizing exogenous DNA,and that form Boer×Chuandong White F₁ goats had a higher ability in internalizing exogenous DNA.The ability in internalizing exogenous DNA of Boer goats was the lowest.The positive sperm rates were(35.99±1.71)%,(32.06±3.21)%and (21.54±2.31)%,which was digested by DNaseâ… ,from the three breeds,respectively.The positive sperm rates from Nanjiang Yellow goat and Boer×Chuandong White F₁ goat were significantly higher than that from Boer goat(P＜0.01).2.There was one SNP(G/A) at the 700 bp of coding region of caprine CD4 gene,which made G234R substitution in the amino acid sequence of caprine CD4 molecule.The Nanjiang Yellow goats had highest hereditary variation than Chuandong White goats,Boer goats,Boer×Chuandong White F₁ goats.3.The SNP of exon 6 of caprine CD4 gene may be an important molecular marker of the ability in internalizing exogenous DNA into sperm of goat spermatozoa.The SNP of exon 6 of CD4 had a significant effect on the ability in internalizing exogenous DNA into sperm of goat spermatozoa(0.01≤P≤0.05).