Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer (SCNT)

// Wei Liu 1, 2 , Li-Hong Wu 1, 2 , Min Yue 1 , Bayaer Nashun 1, 2 , Hua Tang 1, 2 , Yan Chen 1 , Bang-Zhu Chen 1 , Jin Yuan 1 , Dong Xiao 1, 3 and Wei-Wang Gu 1, 2 1 Institute of Comparative Medicine and Laboratory Animal Center, Southern Medical University, Guangzhou 510515, China 2 Pearl Laboratory Animal Sci. and Tech. Co. Ltd., Dongguan 523808, China 3 Guangdong Provincial Key Laboratory of Cancer Immunotherapy Research, Guangzhou Key Laboratory of Tumor Immunology Research, Cancer Research Institute, Southern Medical University, Guangzhou 510515, China Correspondence to: Wei-Wang Gu, email: Guww100@163.com Dong Xiao, email: Xiao_d@hotmail.com Jin Yuan, email: yjin01@126.com Keywords: Dickkopf-related protein 1 (DKK1), lentivirus-mediated gene transfer, somatic cell nuclear transfer (SCNT), Tibet minipigs, transgenic pigs Received: May 09, 2017      Accepted: August 17, 2017      Published: September 01, 2017 ABSTRACT Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.