异种移植血管内皮细胞组织特异性表达人CD59转基因小鼠的建立

Establishment of transgenic mice tissue-specifically expressing human CD59 on the vascular endothclium for xenotransplantation

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 construct transgenic mice tissue-specificaUy expressing hCD59 on the vascular endothelium for xenotransplantation. Methods Transgenic mice were generated by microinjection of a hCD59-minigene under the control of the human intercellular adhesion molecule-2 （ICAM-2） promoter. PCR analysis and Southern blot analysis of genomic DNA were performed to examine the presence of the transgene in the genome of the offspring, and the expression was detected by flow cytometry respectively. Immunohistochemical assay was performed to detect the distribution of hCD59 on the tissues from the transgenic mice. Results After microinjection of gene, 9 of 130 mice born were shown to be transgenic. Human CD59 gene was expressed on the surface of leucocytes in 6 of the 9 hCD59-integrated mice. The expression levels of 6 founders ranged from 80 96 to 95 96 of that on human leucocytes. Human CD59 was strongly expressed on the vascular endothelium of heart, kidney and liver, with little or no positive staining observed on non-endothelial cells. Conclusion The introduced hCD59 gene has been integrated and specifically expressed on the vascular endothelium of transgenic mice.