Construction Of Retroviral Vectors Based On Porcine Endogenous Retroviruses

Xenotransplantation is considered to be a potential solution to overcome the shortage of organs available for transplantation in clinic. So far, pigs are regarded as the most suitable donors. Porcine endogenous retroviruses (PERV) are integrated into porcine germ line DNA as proviruses, and are transmitted vertically. At present, all of the domestic and overseas studies on PERV were focused on the safety evaluation of PERV in xenotransplantation, while there were no reports on applying PERV to produce retroviral vectors. Since PERV are present in the genomes of all pigs, the vector system based on PERV contains homogenous sequences of porcine genome. Thus homogenous recombination may make directed and efficient incorporation of foreign genes possible. The optimization of vector elements may increase expression efficiency greatly. In this study, retroviral vectors were constructed bearing long terminal repeat (LTR) of PERV as transcriptional regulating element. Our studies are described as follows:1 Construction of porcine endogenous retroviral vectorsBased on PERV from Chinese Wuzhishan pigs and Murine Stem Cell Virus (MSCV) vectors, porcine endogenous retroviral vectors were constructed. 5′LTR and 3′LTR of PERV were used to replace the relative domains of MSCV, thus PM-1 vector was construced and named. 5′UTR flanking partial gag and 3′LTR were used to replace the relative domains of MSCV, thus PM-2 vector was construced and named. GFP gene was inserted into Multi-Clone Site (MCS) of PM-1 and PM-2 respectively so that PM-1-GFP-21 and PM-2-GFP-1 expression vectors were constructed. 2 Evaluation of expression system of porcine endogenous retroviral vectorsPK-15 cells were transfected with pPM-1-GFP-21 and pPM-2-GFP-1. By PCR and Southern blotting, it was demonstrated that foreign genes could be delivered and expressed by our vectors successfully. In the parallel, the ability of the vectors to integrate foreign genes to PK-15 genomes stably was confirmed. The packaging status of PM-1 and PM-2 vectors was also analyzed. pPM-1-GFP-21 and pPM-2-GFP-1 were co-transfected with pGag-Pol and pVSV-G respectively into HEK293-T cells. The supernatant was collected to infect PK-15 cells, but expression of GFP gene was not detected. pPM-2-GFP-1 and pVSV-G were co-transfected into PK-15 cells, adding polybrene to promote attachment of virions. However, the result showed no expression of GFP gene.Retroviral vectors PM-1 and PM-2 based on PERV transcriptional regulating elements were constructed successfully in our study. It was also demonstrated that vectors could be integrated into cellular genome DNA stably and foreign genes could be expressed persistently. These results indicated that heterologous LTRs may not contact with packaging plasmids of Gag and Pol. To package the vectors effectively, we should construct its homologous packaging plasmids of Gag and Pol. The results of our study may lead to new method for produce transgenic pigs.