The Technique of Producing HLA-B2704 Gene Transgenic Mice

To produce HLA-B2704 transgenic mice by microinjection,286 Kunming female mice were induced to give superovulation by injecting hormone.The fertilized eggs were collected and microinjected with genomic DNA fragment containing HLA-B2704 DNA into their pronucleus.After microinjection,the survial and two-cell fertilized eggs were implanted into the oviducts of pseudopregnant female mice to let them develop and produce progeny.The intergration of the target gene in F0 and F1 mice was analyzed by PCR.The expression of HLA-B2704 transgene at mRNA level was comfirmed by RT-PCR.8 411 fertilized eggs were collected.6 609 of them were microinjected.The number of survial and two-cell eggs was 4 277 after microinjection.The survial rate was 64.7%.These eggs were implanted into 153 pseudopregnant female mice,26 of which were pregnant and 101 offsprings were obtained at last.PCR analysis showed that genomes of 10 F0 transgenic mice were integrated with the HLA-B2704 gene.The integration rate was 9.9%.Among 78 F1 of positive F0 transgenic mice,15 F1 mice carried the HLA-B2704 gene.HLA-B2704 gene was expressed at the mRNA level in skin,colon,testicle and spleen tissues of the transgenic mice.In the positive HLA-B2704 transgenic mice,six mice were detersile.One mouse showed tumescent at podosoma and toes.Two mice appeared photophobia.One mouse arised diarrhea.The transgenic mice carrying the HLA-B2704 genotype and phenotype were produced successfully.