Screening And Identification Of Wuzhishan Minipigs Inbred Wirh Defective Porcine Endogenous Retrovirus

Organ transplantation is an important treatment for patients with organ failure in the middle and late stages.To solve the problem of clinical donor shortage,people have turned their attention to xenotransplantation.Pigs are very similar to humans in physiological characteristics,organ matching.They have high reproductive rate and stable genetic traits,and are the most suitable donors for xenotransplantation.Porcine endogenous retrovirus(PERV)is present in all pig-derived cells and intact virus particles can be released outside the cell.It was previously integrated into the host genome by provirus and multicopy manner,and cannot be removed by routine breeding of pigs with specific pathogens free(SPF),immunosuppression,and the like.PERV has been shown to infect human cells in vitro and is similar to C retroviruses in integration method,such as Murine leukemia virus(MLV)and feline leukemia virus(FeLV).This has caused people to worry about xenotransplantation induced by zoonosis.The industry guidelines issued by the European Medicines Agency(EMEA)and the U.S.Food and Drug Administration(FDA)have made clear regulations on the ethical issues and pathogenic safety of clinical xenotransplantation.The International Xenotransplantation Association(IXA)has released the list of pathogens for designated pathogen free(DPF)pigs,and describes in detail the conditions under which pigs with islet type I diabetes were tested in clinical trials.In order to make full use of Chinese abundant minipig resources,our laboratory has tested the PERV copy numbers of various minipigs in China in the previous work.Comparing with Guizhou Xiang pigs and Bama miniature pigs,Wuzhishan minipigs has the advantages of lower PERV-A and PERV-B,and no PERV-C type.The inbred line is easy to build a medical model,because of the more than 20 generations of inbreeding and the gene homozygosity of more than 60%.Therefore,we hope to screen out the defective PERV minipigs in the selected low copy of Wuzhishan minipig inbred,and then develop new lines to provide xenotransplantation organ donors.The main work of this study included preliminary screening of the Wuzhishan minipig inbred,confirmation of the experiment to obtain non-PERV infectious pigs,reverse transcriptase activity testing,and one of the pigs for whole genome resequencing.1.Preliminary screening of Wuzhishan minipigs without the PERV contagiousFemoral artery blood of Wuzhishan minipigs was collected.PBMCs were isolated and co-cultured with HEK293 cells for 4 days.PBMCs were removed and total RNA was extracted from HEK293 cells.RT-PCR was used to detect the expression of the structural gene gag,pol and env.Small pigs that do not release the pernicious PERV are candidates.The results showed that 17 out of 105 HEK293 cells had no expression of PERV,and the corresponding minipigs were candidate non-PERV infectious minipigs.2.Confirmation of non-PERV infectious Wuzhishan minipigsThe HEK293 cells co-cultured with the candidate pig PBMCs were cultured for 30 to 60 days.The genomic DNA and total RNA were collected once a week.PCR and RT-PCR were used to detect the new presence of PERV structural genes in HEK293 cells,and filtered out samples that are always negative.The results showed that there were no PERVs present and expression in 12 of the 17 samples.During the culture of HEK293 cells,the supernatant of the cells was collected before each passage,and the reverse transcriptase activity was detected.The OD405 value is less than 2 times the intercept of the vertical axis of the standard curve,indicating that there is no reverse transcriptase activity.If no reverse transcriptase activity was detected at all times,indicating that none of the PERV virus particles were released to the outside,it was verified that the pig was indeed a non-PERV infectious Wuzhishan minipig.The results showed that 8 out of 12 samples could not detect reverse transcriptase activity all the time.The 8 pigs were indeed non-PERV infectious Wuzhishan minipigs.3.Whole genome sequencing of non-PERV infectious pigsOne of the non-PERV infectious Wuzhishan minipigs(body number: 452)with good growth was subjected to whole-genome resequencing.The genomic DNA of PBMC cells was extracted,and detected by the Illumina HiSeq 4000 platform.The selected reference genome was Wuzhishan Inbred pig(Gen Bank: AJKK01000000.1).Quality control of the sequencing data showed that the sequencing was of good quality.The results of sequencing,comparisons with reference genomes,and annotated results of mutation information were counted.All the PERV-pol gene fragments in WZSP452 were found to be incomplete.The only scaffold containing the gag,pol,and env structural genes in WZSP452 was obtained,and all three genes were not fully expressed.4.Cloning and sequence analysis of pol sequence with N basesIn order to obtain the exact sequence information of N base deletion in the pol of the scaffold640,the upstream and downstream primers for the scaffold 640 pol were designed and synthesized.The full length of the scaffold 640 pol was amplified by PCR and connected to the p MD-18 T vector.The recombinant plasmid was transformed into the E.coli DH5?.competent cells.The positive plasmids were identified by colony PCR and double enzyme digestion.The sequencing information is aligned with the whole genome sequence results to obtain complete information for the N bases.We identified a PERV-pol defective pig by performing whole-genome resequencing on one of the selected non-PERV infective Wuzhishan minipigs.This is the world's first time to obtain through inbreeding cultivation combined with natural screening methods,with the advantages of economic and security.The results of this study have promoted the establishment of a new PERV defective new line of Wuzhishan minipigs,which is beneficial to the development and utilization of minipigs in China and provides suitable donors for.