敲除α-1,3-半乳糖基转移酶基因五指山小型猪的繁育和鉴定

摘要

目的 总结 α-1,3-半乳糖基转移酶(GGTA1)基因敲除(GTKO)五指山小型猪的繁育和鉴定.方法 统计GTKO五指山小型猪的繁育结果及窝产仔数;采用聚合酶链反应(PCR)技术对GTKO五指山小型猪GG TA1基因敲除类型进行鉴定;采用荧光显微镜和流式细胞术对人、野生型五指山小型猪、GTKO五指山小型猪外周血单核细胞(PBMC)的α-1,3-半乳糖(αGal)表型进行检测;比较GTKO五指山小型猪与野生型五指山小型猪血常规指标的差异.结果 GGTA 1基因型的遗传符合孟德尔定律;GGTA1-/-猪的PBMC流式检测无荧光表达,与基因型鉴定结果一致;GTKO五指山小型猪雌性猪初产窝产仔数为(6.8±1.8)只,经产窝产仔数为(8.3±2.2)只;血常规检测的各项指标与野生型五指山小型猪差异均无统计学意义(均为P＞ 0.05).结论 连续2代GTKO五指山小型猪遗传稳定,繁殖力正常.GTKO五指山小型猪可作为异种器官移植研究的可靠供体.%To summarize the breeding and identification of Wuzhishan miniature pig models with α-l,3-galactosyltransferase (GGTA1) gene-knockout (GTKO).Methods The breeding and reproduction perform of GTKO Wuzhishan miniature pigs were assessed and the quantity of piglets was counted.The GTKO Wuzhishan miniature pig models with GGTA1gene knockout were validated by polymerase chain reaction(PCR).The αGal phenotype of peripheral blood mononuclear cells (PBMC) in human,wild-type Wuzhishan miniature pigs and GTKO Wuzhishan miniature pigs was detected by fluorescent microscope and flow cytometry.Routine blood test parameters were statistically compared between the GTKO and wild-type Wuzhishan miniature pigs.Results The inheritance of GGTA1 genotype complied with Mendel's law.Flow cytometry detected no fluorescent expression of PBMC in GGTA1-/-pig models,which were consistent with the genotype identification results.The mean piglets of the primiparous GTKO Wuzhishan miniature pigs were (6.8±1.8) and (8.3±2.2) for the multiparous Wuzhishan miniature pigs.No statistical significance was noted in routine blood test parameters between the GTKO and wild-type Wuzhishan miniature pigs (all P＞0.05).Conclusions Stable inheritance and normal reproductive capacity are observed in two generations of Wuzhishan miniature pigs continuously.GTKO Wuzhishan miniature pig is a reliable donor for heterogeneous organ transplantation.