【摘要】 LEA29Y是一种能够高效抑制T细胞活性的新型人工融合蛋白,胰岛表达LEA29Y的转基因猪(Sus scrofa)用于胰岛移植可以阻断T细胞激活的共刺激通路,降低胰岛移植术后人体内T细胞介导的免疫排斥反应,课题组已制备胰岛特异表达LEA29Y的转基因猪。LEA29Y在转基因猪中能否稳定遗传及表达是建立该转基因猪品系的重要鉴定内容,目前尚待明确。本研究连续跟踪2代LEA29Y转基因猪的遗传与表达情况,旨在培育异种胰岛移植的胰岛特异表达LEA29Y转基因猪品系。通过1公(编号81)2母(编号77、83)共3头LEA29Y原代猪(F0代)相互配种或与野生型猪配种进行繁育,得到F1代猪群;F1代阳性个体与野生型猪进行繁育,获得F2代猪群。用PCR对仔猪进行转基因鉴定,反转录PCR(reverse transcriptionPCR,RT-PCR)、免疫组化及免疫荧光染色分析LEA29Y在转基因猪心脏、肝脏、脾脏、肺脏、肾脏、胰腺、肌肉等组织的表达,特别是分析LEA29Y在胰岛的特异性表达。通过繁育建立77、81、83 3个LEA29Y转基因猪支系,繁育正常,共获得95头子代猪,PCR检测53头为阳性。77和83支系中RT-PCR显示F0代77、83LEA29Y表达渗漏;81支系中F0代个体81、F1代个体271 RT-PCR、免疫组化及免疫荧光结果均显示为胰岛特异表达LEA29Y,继代遗传稳定。本研究成功筛选出了遗传稳定的LEA29Y转基因猪品系,可作为异种胰岛移植研究的可靠供体。更多还原

【Abstract】 Exogenous gene stably integrated into the host genome,and stably inherited to the offspring,producing transgenic animals is a basic requirement.Analysis of transgenic animals for inheritance and expression stability of transgene is an important part of establishing a transgenic animal strain.The T cellactivated costimulatory pathway can be blocked by LEA29Y expressed in islet cells of transgenic pig(Sus scrofa),and the T cell-mediated immune rejection in humans after islet transplantation surgery is reduced.A transgenic pig prepared by this research group that specifically expressed LEA29Y in islets,but its inherited genetic condition remains to be determined.In order to obtain islet-specific expression of LEA29Y transgenic pig strain for islet xenograft,the inheritance and expression of two-generation LEA29Y transgenic pigs was continuously traced.1 male(coded 81) and 2 females(coded 77,83),a total of 3 LEA29Y founder pigs(F0)were bred each other or bred with wild-type pigs to get F1 generation pigs.Positive individuals in the F1 generation pigs and wild-type pigs were bred to get F2 generation pigs.The transgenic piglet was detected by PCR.The LEA29Y expression of transgenic pig’s heart,liver,spleen,lung,kidney,pancreas,muscle,and other tissues were tested by reverse transcription-PCR(RT-PCR).Especially the specific expression of LEA29Y in islet was analyzed by immunohistochemistry and immunofluorescence staining.Three founder pigs coded 77,81,83 can all be bred,and a total of 95 progenies were obtained,PCR tested that 53 progenies were positive.RT-PCR results showed that individuals coded 77 and 83 were non-specific expression LEA29Y in 77 and 83 lines.The results of RT-PCR,immunohistochemistry,and immunofluorescence showed that the islet-specific expression of LEA29Y was both in the F0 generation coded 81 and F1 generation coded 271 in 81 lines.And the inheritance of genetic was stable.All above,this study successfully screened for a genetically stable line of LEA29Y transgenic pigs,which can serve as a reliable donor for the study of islet xenotransplantation.更多还原