Construction Of Eukaryotic Expression Vector Of The Key Gene PAX6 In Eye Development And The Establishment Of PAX6/mESCs Cell Line

Objective: To construct an eukaryotic expression vector of the key gene PAX6 in eye and preliminary inquiry the expression in 293 FT cells. PAX6 base was Transfected the PAX6 into mESCs and obtained PAX6/mESCs stable strains, which was the possibility of using PAX6 to induce mESCs to differentiate into LSCs, in order to provide seed cells for corneal damage.Methods:1.Used gene cloning technique to obtain the PAX6 gene and obtained the recombinant plasmid pEF1α-PAX6-IRES-AcGFP, then transfected pEF1α-PAX6-IRES-AcGFP into 293 FT cells with Lipofectamine?2000 assay. The expression of PAX6 protein and GFP were detected by western blot and fluorescence microscope.2.To separating MEF with methods of trypsin digestion in vitro.The growth curve and cell cytoskeleton immunofluorescence compared each generation of MEF. The third generation MEF were dealt with by differentconcentration of Mitomycin C(10μg/ml、20μg/ml and 30μg/ml) at a different time(1h、2h and 3h) to chose the optimal concentration and timing of feeder cells. C57BL/6-mESCs was amplified and cultured in feeder layer. 3. G418 concentration gradient filter to obtain the best screening concentration of mESCs.3.Transfected the vector containing PAX6 into mESCs. For controls, the mESCs were transfected with a pEF1α-IRES-AcGFP vector without PAX6 gene. The mESCs were then screened in the antibiotic G418 to obtain cells that stably expressed PAX6(PAX6/mESCs) or pEF1a-IRES-AcGFP vector without PAX6(pEF1a/mESCs). The transfected cells were identified by PAX6 and GFP immunofluorescence staining. The expression of PAX6 protein in the transfected cells was detected by Western blot. AP. Staining was used to identify the mESCs cells after transfection.Results: 1.A specific band of 1269 bp was detected from recombinant plasmid pEF1α-PAX6-IRES-AcGFP by digestion of Sal I and BamHI and RT-PCR. Sequencing to identify the sequence from GenBank rat PAX6 gene sequence homology of 100%, the size and the direction of the inserted gene were right. After transfection, 293 FT cells showed green fluorescence under fluorescence microscope. A band of PAX6 protein from cells was detected by western blot assay.2.The MEF were obtained by trypsin digestion methed, and the cells showed typical fibroblast morphology, the growth curve determination resulys the 3-5 generation cell proliferation ability better. With technology of CLSM, we observe the change of MEF cytoskelton and the cytoskeleton immunofluorescence staining showed that the 1-3 genetation cell microtubules and microfilaments arranged in neat rows, after 5 generations of cells microtubules and microfilaments disordered cell collapse. Mitomycin C(10μg/ml) 2 hours of feeder cells advantageous to mESCs cells culture. 3. 200 μg/ml G418 for mESCs optimal screening concentration; After transfected with recombinant plasmid, cytoplasm green fluorescent protein expression was significantly, Western blot results showed that PAX6 in PAX6/mESCs expressed protein. After transfection of mESCs morphology to the characteristics did not change, the performance of AP staining was positive in the over expression of exogenous protein PAX6.Conclusion: This experiment successfully constructed recombinant plasmid pEF1α-PAX6-IRES-AcGFP and expression in 293 FT cells; Successfully isolated and cultured and amplified the MEFs to use as feeder layer for mESCs cultured. Recombinant plasmids by liposome transfection method can be PAX6 transfected into mESCs and G418 selection PAX6/mESCs steady strain. After transfection of mESCs in the expression of the PAX6 and maintain the stem cells can, for further study of PAX6 induced differentiation of mESCs lay a good foundation.