Production Of Transgenic Pigs Expressing Human CD46 And Its Function Analysis

Using genetic engineering techniques, transgenic and somatic cell cloning technology this study constructed a human CD46 gene expression vector and generated transgenic wuzhishan miniature pigs with a high level of hCD46 expression. Functional analysis of the transgene was also performed in vitro. The following results were obtained:1、Gene regulatory sequences of the CD46 was acquired by gene-capture technology, and cDNA sequences of the human CD46 gene was synthetical. The splice of the two sequence composed the transgene construct,with adding of a resistance marker genes for selection of the transgene positive cells; the expression of the transgene construct was confirmed by transfecting fibroblasts cells.2、Primary fibroblast cells of Wuzhishan mini-pigs were builded. AMAXA Nucleofector was used to transfer vectors to builded fibroblasts cells; 48h after transfection, puromycin was added to the medium for selection of transgene positive cells; obtained cells were identified by RT-PCR and stained by FITC labeled antibody.3、The selected positive cells was used for somatic cell nuclear transfer and embryos transfer.1260 embryos was transferred to surrogates 6,5 of which was pregnant. Finally,3 surrogates successly deliver litter 11 piglets. Preliminary identification of the transgene showed 10 positive piglets.4、Expression of hCD46 with a distinct tissue-specific pattern similar to the human one was observed using immunohistochemistry. Various tissues revealed strong immunostaining in most epithelial tissues and vascular endothelium.5、Flow cytometry analysis of hCD46 aortic endothelial cells (AECs) showed that high levels of hCD46 expression was acquired compared to human AECs. The expression of AECs was higher than fibroblast cells of the transgene pig.6、An in vitro study showed a protective effect of hCD46 on porcine AECs against human complement-mediated cytolysis. Distinct differences between ABO blood groups were also observed, with much less cytolysis of porcine AECs treated with B blood group serum than with other serum types.The present study has obtained high level hCD46-expressing transgenic pigs on an inbred Wuzhishan miniature pig background. Complement-mediated cytolysis of transgenic porcine AECs with human serum showed significant protection. These animals will provide valuable donors for preclinical studies in xenotransplantation of pig tissues to nonhuman primate.