Construction Of Recombinant Porcine PLL-Myc And PLL-Klf4 Lentiviral Vector

Recently, to avoid controversy of Ethics, scholars at home and abroad introduced four transcription factors, which are Oct4, Sox2, c-Myc and Klf4, to the mouse skin fibroblasts through the vector. This method could reprogram the mouse skin fibroblasts, embryonic stem cell was produced by this method, which is similar to the pluripotent cells, and pluripotent stem cell lines (induced pluripotent stem cells, iPS cells)also could be established .According to the c-Myc and Klf4 gene sequences known, c-Myc and Klf4 primer were designed and synthesized. The c-Myc and the Klf4 gene fragments were obtained using reverse transcriptase polymerase chain reaction (RT-PCR). The tatal RNA was extracted from the fetal pig genital ridge, and reverse transcription to cDNA, pig c-Myc and Klf4 genes was amplified by PCR. The c-Myc and Klf4 genes was recovered and purified; and then digested by the restriction enzyme HindⅢ,BamHⅠ, T vector was connected to c-Myc and Klf4 gene fragments respectively by T4 ligase, and the product was transformed by competence-DH-5αbacteria and then screened by Amp, the bacteria lipid was identified by PCR and sequenced. The result showed that the c-Myc and Klf4 genes were the same with the known gene sequences. The target gene fragment and pLEGFP-N1 vector digested by the restriction enzyme HindⅢ,BamHⅠ, pLEGFP-N1 vector was connected to c-Myc and Klf4 gene fragments respectively by T4 ligase, and the product was transformed by competence-DH-5αbacteria and then screened by Amp, the bacteria lipid was identified by PCR. The result showed that retroviral expression vector pLEGFP-Myc and pLEGFP-Klf4 were constructed successfully. The pLEGFP-Myc, pLEGFP-Klf4, and pLentiLox3.7 vectors enzyme digested by the restriction endonuclease enzyme. The product was recovery and purified. The pLentiLox3.7 vector was connected to Myc-GFP; Klf4-GFP respectively by T4 ligase, and the product was transformed by competence-DH-5αbacteria and then screened by Amp, the bacteria lipid was identified by PCR. The result showed that retroviral expression vector pLL-Myc, pLL-Klf4 were constructed successfully. Mini Preparation was from the recombinant bacterial plasmid, and a quality of recombinants of pLL-Myc, pLL-Klf4 which were infected with viral vector-assisted 293FT packaging cells were obtained. Then cells were cultured for 24 hours, and under the inverted fluorescence microscope, the expression of green fluorescent protein was observed. The purpose of this experiment is that constructing lentiviral expression vectors pLL3.7-Myc and pLL3.7-Klf4 of porcine. Aim to establish the base on the study of iPS cells and effects of c-Myc and Klf4 gene to the biological development.