Construction Of RPNCE-Lenti1259 Recombinant Lentiviral Vector And Transfecting Human Embryonic Stem Cells

Embryonic stem cells (ESCs) are totipotent stem cells, with strong self-renewal capacity and multilineage differentiation potential. 1998, Thomson and his colleagues successfully separate and set up the first human embryonic stem cell lines. Human ES cells are induced to differentiate into pancreatic precursor cells, which provide a rich source of cells for diabetes' cell replacement therapy. But it has not been an ideal way to separate pancreatic precursor cells from the mixed cultures. In order to overcome this difficulty, we took the following studies:1. Expression and evaluation of pPDXA12-EGFP-C3 recombinant vector, gene transfection in vitro and analysis of transgenic vectors. PDXA12 promoter was amplified by polymerase chain reaction (PCR) from human genomic DNA template. AseI enzymed cite was inserted in to 5' of forward primer, and NheI enzymed cite was inserted into 5' downstream primer. Then production of PCR was ligated with pMD19-T vector. Using the DNA recombinant techniques, the eukaryotic expression vector pPDXA12-EGFP-C3 recombinant vector was constructed, and PDXA12 promoter inserted insense orientation were confirmed and by restriction endonuclease digestion analysis. The pPDXA12-EGFP-C3 recombinant vector was transferred into NIT-1, HEFS, 293FT, and hESCs cells. The NIT-1 cell lines with pPDXA12-EGFP-C3 recombinant vector transient transfection were investigated under fluorescence microscope, and the expression of enhanced green fluorescent protein could be observed. Reverse transcriptase PCR was used to detect the expression of EGFP gene at the level of messenger RNA (mRNA) and so did inununohistochemical method to test the expression of EGFP gene. Results showed the target gene could express correctly in host cells. These identified vectors were also transferred into HEFS, 293FT, and hESCs cells. The target gene couldn't express in HEFS, 293FT, and hESCs cells. These experiments prove that pPDXA12 promoter could specifically guide EGFP gene expressing in pancreatic islet cells express.2. Expression and evaluation of rPNCE-Lenti1259 lentiviral vector, gene transfection in vitro and analysis of transgenic vectors. PDXA12 promoter was amplified by polymerase chain reaction (PCR) from pPDXA12-EGFP-C3 recombinant vector. XbaI enzymed cite was inserted in to 5' of forward primer, and XmaI enzymed cite was inserted into 5' downstream primer. Then production of PCR was ligated with pMD19-T vector. Using the DNA recombinant techniques, the eukaryotic expression vector rPNCE-Lenti1259 lentiviral vector was constructed, and PDXA12 promoter inserted insense orientation were confirmed and by restriction endonuclease digestion analysis. The rPNCE-Lenti1259 lentiviral vector was transferred into NIT-1, HEFS, 293FT, and hESCs cells. The NIT-1 cell lines with rPNCE-Lenti1259 lentiviral vector transient transfection were investigated under fluorescence microscope, and the expression of enhanced green fluorescent protein could be observed. Reverse transcriptase PCR was used to detect the expression of Neo~r gene at the level of messenger RNA (mRNA) and so did inununohistochemical method to test the expression of Neo~r gene. Results showed the target gene could express correctly in host cells. These identified vectors were also transferred into HEFS, 293FT, and hESCs cells. The target gene couldn't express in HEFS, 293FT, and hESCs cells. These experiments prove that PDXA12 promoter could specifically guide Neo~r gene expressing in pancreatic islet cells express.3. Lentiviral vector successfully transfecting human ES Cell. Using rPNCE-Lenti1259 lentiviral vector; CMVAR8.91; MD.G (expressing vesicular stomatitis virus glycoprotein, VSVG-seudotyped) were transferred together into 293FT cells, the viral supernatant is ready for collection 60 hours after transfection. Adjust centrifuge to spin for 90 minutes at 4℃, 25000 rpm. When spin is finished, remove all traces of supernatant, then add 100 ulof cold PBS, keep tubes at 4℃for at least 12 hours to dissolve the pellet. Human ES cell culture conditions are relatively harsh, not easy to exogenous gene transfection. In order to improve the lentiviral infection efficiency of human ES cells, coating the plates with poly-d-lysine, changing the packaging cell density, selecting suitable medium to collectvirus supernatant were used in this experiment. At last, the lentiviral transient transfection efficiency was about 30-40%. Due to the high efficiency of lentiviral stable integration, human ES cell clones stably transfecting rPNCE-Lenti1259 lentiviral vector could be screened by EGFP marker.In this study, we construct the vector using specific pancreatic issue promoter PDXA12 carrying the Neomycin gene driven by a specific pancreatic tissue expressing promoter, To construct recombinant pPDXA12-EGFP-C3 vector and rPNCE-Lenti1259 lentiviral expression vector. The recombinant constructed vector was verified and proved that PDXA12 promoter can effectively guide the target gene expression in islet cells at the cellular and molecular level. Human ES cell lines stably transfected PDX1-specific neomycin resistance gene were established by using pancreatic-specific promoter and neomycin resistance gene of recombinant lentiviral vector system. This study has laid a good experimental foundation for establishing human ES cell clones with stably transfecting rPNCE-Lenti1259 lentiviral vector and purifying pancreatic precursor cells.