The Screening And Identification Of PERV Free CEMPs & The Construction Of Pig MSTN Knock-out Vector

Part one The Screening and Identification of Endogenous Retrovirus Free CEMPsThe endogenous retrovirus (ERV) is one kind of retrovirus that integrates in the genome of host cells in the form of provirus and replicates with the proliferation of host cells, and ERV itself is not pathogenic to its hosts. It is by estamination that ERV integrates into host cells and settles there about 120 million years ago, and ERV may play an important role in the evolution, pathology and physiology of animals. By now, nearly all mammalians including human beings contain ERV in their genome.The porcine endogenous retrovirus (PERV) is a gamma-retrovirus of type C that integrates in the genome of pigs and transmits vertically131. Studies showed that PERV descends from the same branch of the phylogenetic tree as gibbon-ape leukemia virus (GALV), murine leukemia virus (MLV), and feline leukemia virus (FeLV). From the context of genetic distance, it was estimated that the viruses integrated into the pigs' genome about 30 million years ago.Pigs have many advantages to be organ donors for xenotransplantation, but the PERV contained in their genome and carbohydrate antigen the Gal-a -1,3-Gal distributed in the surface of cell membrane stands in the way to be the major barrier that restricts pigs as organ donors. Researches have shown that the copy number of PERV varies greatly among different breeds and individuals, and the copy number of PERV in some pigs such as the Xiang pigs is very low151, some breeds just have none of PERV, as in the South America peccaries'61.In the experiment, 67 healthy, female Chinese Experimental Mini-Pigs (CEMPs) aged at 3 to 6 months were selected as subjects from the Animal Husbandry Station of China Agricultural University. First, the copy numbers of PERV distributed in the genome of CEMPs are investigated through large-scale screening by PCR analysis, two pigs were initially found to be negative. Then, in order to further ascertain our results, Southern blotting was employed for verification, and the results are in accordance with that acquired from PCR analysis.During the research, 40 CEMPs and Landrance containing PERV were also selected respectively to analyze the envelope gene types. It is showed that the distribution of types of envelope protein gene in Landrace and CEMPs makes little difference, but the proportion of individuals carrying two types of envelope protein gene (env-A and env-B, which is denoted as env-AB) is much larger than those whichcarry only one type of envelope protein gene (env-A or env-B).By using the methods of PCR and Sounthern blotting, two endogenous retrovirus free pigs were found for the first time during our research, and the copy number of others is relatively low, which is about 10 to 20. All the results illuminate the genetic diversity of indigenous pig breeds in China and the potential of CEMPs to serve as organ donors in xenotransplantation. Meanwhile, this also provides the basic materials of using CEMPs as organ donors in xenotransplantation.