摘要
目的:建立携带人α1,2-岩藻苷转移酶[alpha(1,2)-fucosyltransferase,HT]基因的转基因小鼠模型。方法:采用受精卵显微注射法,将人HT转基因构件导入昆明白小鼠受精卵的雄原核内,然后将注射后仍健康的受精卵植入假孕母管内,等其自然分娩。对G0代小鼠,应用聚合酶链反应(PCR)和Southern杂交检测人HT基因的整合情况、应用逆转录PCR(RT-PCR)检测人HT基因mRNA水平的表达,应用流式细胞计数(FCM)检测H抗原及α-Gal抗原在小鼠外周血单核细胞(PBMCs)表面的表达。结果:注射后卵的存活率和幼鼠出生率分别为84.9%(968/1140)和10.8%(105/968),外源基因的整合率为7.6%(8/105),基因整合阳性小鼠中有7只的心、肝、肾及骨骼肌组织均有人HTmRNA表达,并且PBMCs表面H抗原表达阳性,表达强度为人PBMCs的95%～150%,同时α-Gal抗原表达减少,为正常小鼠的5%～10%。转基因小鼠已经稳定传3代。结论:携带人α1,2-岩藻糖苷转移酶基因的转基因小鼠模型已制备成功。
Objective： To generate transgenic mice model of human alpha （1,2）-fucosyltransferase（HT） gene. Methods： The transgenic mice were produced by microinjection of fertilized ovum and transgene construction containing human HT gene was implanted into male pronucleus of zygoto of Kunming white mice then the injected healthy fertilizered egg was implanted into uterine tube of pseudocyesis female mice and waited for natural delivery. The integration of human HT gene in the Go generation mice was tested by polymerase chain reaction （PCR） and Southern blot, mRNA expression of human HT gene was detected by reverse transcription ploymcrase chain reaction （RT-PCR） analysis. The expression of H antigen and α-Gal antigen in peripheral blood mononuclear cells （PBMCs） in mice were detected by Flow Cytometry （FCM）. Results： The ovum survival rate after microinjection and the birthrate of offsprings were 84.9% （968/1140） and 10.8% （105/968） respectively. Integration rate of human exigenous HT gene was 7.6% （8/105）. Seven mice in the positive gene integration group had a human HT mRNA expression in the heart, liver, kidney and muscle. What is more, expression of H antigen whose intensity was 95%-150% of human was positive on PBMCs. Meanwhile, reduction of α-Gal antigen on PBMCs in mice was 5%-10% of normal mice. The human HT gene was steadily transferred to the third generation. Conclusion： The transgenic mice model of human HT gene has been produced successfully.
出处
《天津医药》
CAS
北大核心
2006年第10期708-710,754,共4页
Tianjin Medical Journal
基金
天津市科技发展计划项目重点资助(项目编号:043803411)
