Generation Of DKK1Transgenic Tibet Minipigs By Somatic Cell Nuclear Transfer (SCNT)

Background:Because of small size of body, easy to operate, and anatomical, physiological and biochemical similarites to humans, at present minipigs are becoming the most popular used large laboratory animals.In2004, Professor Gu Weiwang (Laboratory Animal Center, Southern Medical University) introduced Tibetan pigs to Guangzhou for acclimatization; it took5years to breed Tibetan pigs into a new experimental miniature pigs, named Tibet minipigs.As the structure of porcine and human skin is very similar, pigs are considered an ideal model for studies related with skin. Pigs are widely used in researches like skin grafting, cosmetic identification, ultraviolet radiation, skin cance, burns, frostbite, skin aging and etc. However, because of pig skin coat, shaving process is inevitable before skin test or surgery.Commonly used small laboratory animals without hair have nude mice, Yuyi hairless mice and hairless guinea pigs; hairless guinea pigs have been widely applied in skin-related researches (such as hair tonic effect, skin allergies, skin grafting treatment, ultraviolet radiation response, etc). Currently, Yucatan miniature pig is the world’s only hairless pig strain, and widely used in skin research. At present, China has nurtured many miniature pig strains, including:Wuzhishan miniature pigs, Guizhou miniature pigs, Bama miniature pigs, Banna miniature pigs and Tibet minipigs, etc, but not yet bred hairless miniature pig breeds. Biomedical researches yearn for the appearance of hairless miniature pigs.Hairless Tibet minipigs can simplify the hair removal procedures before experiment and avoid the skin damage caused thereby. Tibet minipigs can be widely used in researches like skin grafting, cosmetic identification, ultraviolet radiation, skin cance, burns, frostbite, skin aging etc., and provide a good animal model for alopecia research, which makes it have great significance in biomedical researches.In mice, the skin-specific overexpression of DKK1transgene under control of human skin K14promoter in transgenic mice led to hairless in mice, suggesting that hairless mimipigs might be attained by the skin-specific overexpression of DKK1transgene in transgenic pigs.Against this background, we will intend to generate the DKK1transgenic Tibet minipigs by somatic cell nuclear transfer (SCNT) to create hairless mimipigs by the skin-specific overexpression of DKK1transgene in transgenic pigs.Methods1. Construction of lentiviral vector of pERKDZG carrying DKK1gene of Tibet minipigsTo create hairless mimipigs, we firstly construct lentiviral vector of pEFla-RFP-K14-pDKK1-IRES-ZsGreen (pERKDZG) by standard molecular biology methods. Among pERKDZG, the expression of red fluorescent protein (RFP) gene is regulated under control of EF1α promoter, while the expression of pig DKK1gene (pDKK1) and green fluorescent protein (ZsGreen) gene is regulated under control of human skin K14promoter.First, total RNA is extracted from liver tissue of Tibet minipig, and then reverse transcribed into cDNA, followed by amplifying the coding region of Tibet minipig DKK1(abbreviated as pDKK1) gene. After that, pERKDZG will be generated by the following3-step DNA cloning:1) pDKKl gene is inserted into the vector pK14-DKK1to replace the fragment of mouse DKK1to finally obtain the vector of pK14-pDKKl;2) The fragment of EFla-RFP which is amplified from pCDH-CMV-MCS-EFla-RFP is cloned into the lentiviral vector of pHAGE-fullEFla-MCS-IzsGreen to pEREZG;3) The fragment of K14-pDKK1which is amplified from pK14-pDKKl is inserted into pEREZG to finally obtain pERKDZG.2. Generation of DKK1transgenic Tibet minipigs of by somatic cell nuclear transfer (SCNT)The lentiviruses produced by using the lentiviral vector of pERKDZG infect PEFs, followed by RFP and GFP assay. RFP-positive PEFs will be sorted out by flow cytometry (FCM) to obtain the purified PEFs carrying ERKDZG (referred to as PEF-DKK1used for somatic cell nuclear transplantation). PEF-DKK1is employed to generate DKK1transgenic Tibet minipigs of by somatic cell nuclear transfer (SCNT) to attain hairless pig.Results: 1. Construction of lentiviral vector of pERKDZG carrying DKK1gene of Tibet minipigsThe DKK1gene of Tibet minipigs was successfully cloned, while the successful construction of lentiviral vector of pERKDZG is confirmed by restriction enzyme and sequencing.2. Generation of DKK1transgenic Tibet minipigs of by somatic cell nuclear transfer (SCNT)The K14-pDKKl transgene is transferred into PEFs by lentivirus-mediated gene transfer, followed by obtaining the purified PEFs carrying ERKDZG (referred to as PEF-DKK1) via sorting out by using flow cytometry (FCM). PEF-DKK1is employed to generate DKK1transgenic Tibet minipigs of by somatic cell nuclear transfer (SCNT). The blastocyst rate is about20%.7days after in vitro culture, the reconstructed embryos develop well and normally express RFP, indicating the normal expression of exogenous transgenes in reconstructed embryos. Reconstructed embryos were transplanted into5recipients, while2recipients are pregnant, and8cloned piglets are born, in which3have DKK1transgene and successfully express RFP and GFP.Conclusion:1. Successful construction of lentiviral vector of pERKDZG carrying DKK1gene of Tibet minipigs;2. Successful preparation of PEF-DKK1used for somatic cell nuclear transplantation;3. By somatic cell nuclear transfer (SCNT), cloned piglets carrying the Tibetan minipig DKK1transgene were successfully attained.