利用细菌人工染色体构建人SIRPα转基因兔模型  

Construction of rabbit model expressing human SIRPαgene using bacterial artificial chromosome

作　　者：杨素杰 陶源 耿阳 赵多维 李传沛 尹智[1] 赵祥杰 刘忠华[1] 宋军[1] YANG Sujie;TAO Yuan;GENG Yang;ZHAO Duowei;LI Chuanpei;YIN Zhi;ZHAO Xiangjie;LIU Zhonghua;SONG Jun(School of Life Sciences,Northeast Agricultural University,Key Laboratory of Animal and Cell Genetic Engineering of Heilongjiang Province,Harbin 150030,China)

机构地区：[1]东北农业大学生命科学学院,黑龙江省动物与细胞遗传工程重点实验室,哈尔滨150030

出　　处：《东北农业大学学报》2024年第8期171-180,共10页Journal of Northeast Agricultural University

基　　金：国家自然科学基金项目(32070540)。

摘　　要：异种细胞和组织移植是免疫缺陷动物主要研究方向之一,为增强免疫缺陷动物对人源细胞与组织的亲和性,降低免疫排斥反应,常通过转入人SIRPα(Signal regulatory proteinα)基因使其免疫细胞表达人源SIRPA蛋白。hSIRPA蛋白可识别并结合人源移植细胞上的CD47分子,形成“不要吃我”信号,抑制吞噬细胞吞噬,显著降低受体动物对外来细胞和组织的免疫排斥反应,提高其在动物体内的移植效果。研究通过显微操作技术将含有hSIRPα基因的BAC质粒注射到兔受精卵原核中,经胚胎移植成功获得h SIRPα转基因兔。通过PCR、real-time PCR、流式细胞术等方法检测转基因兔基因型与表型。结果表明:通过检测F0和F1两代hSIRPα转基因兔基因型,hSIRPα基因被完整地转入兔体内。在转基因各组织中hSIRPα基因表达水平存在差异,在回肠、胃、脾脏中,hSIRPα基因表达水平显著高于野生型兔;外周血流式细胞技术检测结果可知,hSIRPα在转基因兔的表达水平高于野生型,不影响兔免疫细胞及组分正常发育。综上,试验成功获得hSIRPα转基因兔,为获得类似于NSG、NOG小鼠的免疫缺陷兔模型奠定基础。Xenogeneic cell and tissue transplantation is one of the main research directions in immunodeficient animals.In order to enhance the affinity of immunodeficient animals to human cells and tissues and reduce immune rejection,human signal regulatory proteinαgene is often transferred into immune cells to express human SIRPαprotein.hSIRPA protein can recognize and bind to the CD47 molecule on human transplanted cells,forming a"Don't eat me"signal,causing the phagocytosis of phagocytes to be inhibited,significantly reducing the immune rejection of foreign cells and tissues in recipient animals,thereby improving its transplantation effect in animals.In this study,the BAC plasmid containing hSIRPαgene was injected into the pronucleus of rabbit fertilized eggs through micromanipulation technology,and then transplanted the injected fertilized eggs into the fallopian tube of the recipient rabbit through embryo transfer,and successfully obtained hSIRPαtransgenic rabbits.The genotype and phenotype of transgenic rabbits were detected by PCR,real-time PCR,flow cytometry and other methods.The results showed that through the hSIRPαgene was completely transferred into the rabbits the genotype detection of hSIRPαtransgenic rabbits in the F0 and F1 generations There were differences in the expression levels of hSIRPαgene in various transgenic tissues.In the ileum,stomach and spleen,the expression level of hSIRPαgene was significantly higher than that of wild-type rabbits;the flow cytometric detection results of peripheral blood showed that hSIRPαwas expressed in transgenic rabbits,and the expression level was higher than that of wild type,which could not affect the normal development of rabbit immune cells and components.In summary,the experiment successfully obtained hSIRPαtransgenic rabbits,laying the foundation for obtaining an immunodeficient rabbit model similar to NSG and NOG mice.

关 键 词：细菌人工染色体(BAC) 转基因兔模型 h SIRPα基因 SIRPα-CD47抑制性信号 

分 类 号：S829.1[农业科学—畜牧学] Q78[农业科学—畜牧兽医]