Construction Of Transgenic Mini-pigs With Kidney Specifically Expressing Cre Recombinase

Cystic kidney diseases, were kind of autosome dominant disease and very common in clinical practice. These diseases were life-threatening in human. Currently, the animal models were used to investigate the pathogenic mechanism of human cystic kidney diseases, such as mouse models. However, the characteristic of anatomy and physiology was significantly different between mice and human. Thus, mouse models which were gene modification was not appropriate to replication the process of human cystic kidney diseases. In contrast to mice, the feature of organ and physiology was similar to human in the pig model. Therefore, the pig model was more favourable for research of human diseases in scientist at this point.Aquaporin, a membrane protein, involve in water transport efficiently. Aquaporin 2, was the member of Aquaporin and expressed in kidney’s collecting duct cells in mammals. In this study, the 8 kb of Aquaporin 2 promoter was used to drive to Cre expression in transgene pig. The Cre-loxp system was the common conditional gene targeting strategies which were dependent on the expression of Cre. Compare with different cells of porcine, our results suggested that the p ET28a-AQP2-Cre plasmid specificity expressed in the cells of kidney. Furthermore, fetal fibroblasts were transfected by p ET28a-AQP2-Cre plasmid as the nuclear donor cells. Using somatic cell nuclear transplantation, reconstructed embryos which carried p ET28a-AQP2-Cre gene were transplanted in surrogate sow. Finally, two transgene mini-pigs were harvested in our study.Our results of RT-PCR and Western blotting suggested that the Cre specificity expressed in the kidney of transgene mini-pigs, while no expression was observed in control tissues. The immunohistochemical results also revealed that Cre specificity expressed in the collecting duct cells of transgene porcine kidney and no expression in control. Thus, our results indicated that the Cre was specificity expressed in kidney of p ET28a-AQP2-Cre transgene pigs.In our study, the q PCR and hi Tail-PCR were used to invested copy number of Cre and integration site analysis, respectively. Our q PCR result suggested that the Cre copies were 12- 14 in diverse tissues in p ET28a-AQP2-Cre transgene pigs. The results of hi Tail-PCR revealed that six integration sites of p ET28a-AQP2-Cre were identified.Based on our data and combined with previous studies, we believe that the p ET28a-AQP2-Cre transgene pigs not only play an important role in the knockout gene of collecting duct cells, also have the crucial role in the research on the pathogenic mechanism of human cystic kidney diseases.