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目的建立用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。方法采用受精卵显微注射技术,将含有人ICAM-2启动子、人CD59基因第1个内含子、人CD59 cDNA、BGH polyA终止信号的外源基因导入小鼠受精卵的原核中,选取注射后仍健康的受精卵移植入假孕母鼠的输卵管中待分娩。聚合酶链反应(PCR)及Southern blot确定外源基因整合阳性转基因小鼠。流式细胞术用于外源基因蛋白质水平表达检测。免疫组织化学方法观察人CD59在转基因小鼠心脏、肝脏、肾脏等器官表达分布情况。结果产仔130只,9只外源基因整合阳性,整合率6％(9／130)。6只获得蛋白质水平表达,蛋白质水平表达强度为人CD59在人白细胞表达强度的80％至95％,转基因效率5％(6／130)。免疫组织化学检测显示人CD59在转基因小鼠心脏、肝脏、肾脏组织有较强表达,且表达限于血管内皮细胞。结论成功地建立了用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。
Objective To establish a tissue-specific human CD59 transgenic mouse for xenotransplantation studies in vascular endothelial cells. Methods The exogenous gene containing human ICAM-2 promoter, the first intron of human CD59 gene, the human CD59 cDNA and the termination signal of BGH polyA was introduced into the pronucleus of mouse zygotes by microinjection of zygotes. Fertilized eggs that are still healthy after injection are transplanted into the fallopian tubes of fake pregnant rats to be delivered. Polymerase chain reaction (PCR) and Southern blot were used to confirm the foreign gene integration positive transgenic mice. Flow cytometry is used for the detection of exogenous gene protein expression levels. Immunohistochemistry was used to observe the expression of human CD59 in the heart, liver and kidney of transgenic mice. Results 130 litters and 9 foreign genes were positive for integration, the integration rate was 6% (9/130). 6 obtained protein expression level of protein expression level of human CD59 in human leukocyte expression intensity of 80% to 95%, transgene efficiency of 5% (6/130). Immunohistochemistry showed that human CD59 was strongly expressed in the heart, liver and kidney tissues of transgenic mice and the expression was restricted to vascular endothelial cells. CONCLUSIONS: Human CD59 transgenic mice that have been specifically engineered for xenograft vascular endothelial cell tissue have been successfully established.