Cloning and pluripotency identification of Nanog gene in Rongchang pig
[Objective]The Nanog gene is a transcription factor that regulates the maintenance of stem cell pluripotency,and ectopic expres-sion of Nanog can lead to some pluripotency in somatic cell reprogramming.Cloning the Nanog gene of Rongchang pigs,constructing a pMX retroviral vector,and identifying the transfection efficiency of the expression vector will lay the foundation for further genetic improvement and stem cell research of Rongchang pigs.[Method]Using the reproductive crest of Rongchang pigs as material,total RNA was extracted and re-verse transcribed into cDNA.The Nanog gene of Rongchang pigs was amplified and cloned by PCR.The ear tissue from Rongchang pigs and culture Rongchang pig fibroblasts were colleeted using tissue block method.The 3rd to 5th generation of pig fibroblasts were selected as trans-fected cells,and the pMD18T-Nanog vector was constrcuted through T-A cloning;The Nanog gene was ligated to the pMX retroviral vector by T4 ligation method,and the pMX-Nanog-neo expression vector was constructed after enzyme digestion identification.Then,the reporter gene EGFP was ligated to the pMX-Nanog-neo to construct the pMX-Nanog-EGFP expression vector.The liposomal LTX method was used for trans-fection,and pMX-Nanog vector was mixed with pCMV-VSVG in the ratio of 2∶1.Then the pMX-Nanog-EGFP expression vector was allowed to transfect 293 GP packaging cells and the viral supernatant was collected for spare parts,and Hoechst-stained nuclei were used for immuno-histochemical identification of its transfection and expression effects with Nanog antibody.Subsequently,the pMX-Nanog-neo expression vector was transfected into Rongchang pig fibroblasts amplified by the same method,and the positive cell lines were also screened by G418.[Re-sult]The Nanog gene of Rongchang pig was successfully cloned,and pMX-Nanog-neo and pMX-Nanog-EGFP reverse transcription vectors were successfully constructed.The pMX-Nanog-EGFP expression vector was transfected into 293GP cells,and stable expression of EGFP fluo-rescence was visible after 48 hours.After cell expansion and culture,the Nanog gene was confirmed to be a stable nuclear expression protein through Hoechst staining and Nanog antibody immunohistochemistry identification.Thus,it was confirmed that the constructed retrovirus vec-tor could efficientively transfect cells and stably express the target genes.Porcine fibroblasts were transfected with pMX-Nanog-neo vector,and the positive cell lines stably expressing Nanog gene were obtained by G418(600 ng/mL)screening.[Conclusion]The constructed pMX ret-rovirus expression vector can stably transfect the target gene Nanog into Rongchang pig fibroblasts and efficiently express it,which will pro-vide a reliable technical method for study on the pluripotency maintenance mechanism,self-renewal mechanism,differentiation mechanism and reprogramming mechanism of Rongchang pig stem cells in the future.
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维普
