携带HLA-B2704基因转基因小鼠技术的建立

应用显微注射法制备携带HLA B2 70 4基因的转基因小鼠 .对 2 86只昆明小鼠激素注射进行超排卵 ,采集受精卵 ,将含HLA B2 70 4基因的基因组DNA片段 (简称HLA B2 70 4DNA)显微注射到受精卵原核内 ,把注射存活的两细胞期受精卵移入假孕鼠的输卵管内使其发育产生后代 .用PCR方法进行F0代仔鼠及F1代仔鼠的转基因整合的检测 .利用RT PCR检测阳性鼠中的HLA B2 70 4转基因的表达 .采集了 84 11个卵 ,可注射卵 6 6 0 9个 ,其中注射存活的两细胞期受精卵 4 2 77个 ,卵的注射存活率为 6 4 7%.将卵移入 15 3只假孕鼠 ,其中 2 6只怀孕产仔 ,存活 10 1只 .在 10只F0代仔鼠基因组中有HLA B2 70 4基因整合 ,整合率为 9 9%.转基因阳性鼠F0代之间以及与正常鼠之间进行交配 ,产生的F1代仔鼠 78只 ,其中 15只为阳性 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有HLA B2 70 4转基因mRNA的表达 .在HLA B2 70 4转基因阳性小鼠中 ,6只小鼠皮肤出现脱毛 ,1只小鼠的足部及足趾明显红肿 ,2只在脱毛同时明显畏光 ,1只出现腹泻 .结果表明 ,成功地建立了HLA B2 70 4的转基因小鼠技术 ,该小鼠类似强直性脊柱炎的小鼠模型 .

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.