The Study Of Construction And Packing Of Adenovirus Vector-Oct4 Sox2、C-Myc、Klf4 And Infecting Mouse Embryonic Fibroblast

Background:Induced pluripotent stem cells (iPSCs) were reprogrammed from somatic cells using specinc transcription factors.Bypassing the ethical issue caused by embryonic stem cells(ESCs), iPSCs can be successfully induced from a variety of cells, which makes iPSCs a powerful research tool for developmental biology.we review the research progress of increasing the reprogramming mechanism and genetic stability of iPSCs in order to provide references of reprogramming eciency of iPSCs, reducing the cost, and addressing key points of iPSCs quality control, further promoting clinical application of the iPSCs. Induced pluripotent stem(iPS)cells were produced by forced expression of four transcription factors-Oct4, Sox2, Klf4 and c-Myc in differentiated somatic cells, this revolutionized approach has attracted great scientific and public attention, and the generation of iPS cells from individual patients has raised hopes for treating many degenerative and genetic diseases as this technique could circumvent the ethical conflict and avoid immune rejection after transplantation which should be faced in ESCs application. Objective:To construst and pack adenovirus vector which includes the four genes as Oct4, Sox2, Klf4 and c-Myc, restructuring can at the same time the four factor of Adenovirus, and infect primary cultured mouse embryonic fibroblast (MEF).Mainly to illustrate construction and packing of adenovirus vectors and investigate the optimum condition of target gene over-xpression lentivirus particle infecting mouse embryonic fibroblastcells. Methods:First:To acquire objective genes from the plasmid which is Oct4, Sox2, Klf4 and c-Myc. Cut aim genes and enzymes linearization of adenovirus vector orientation connection, using enzyme digestion and polymerase chain reaction (PCR) to identify the correctness of the viral vector, to grow a positive cloning sequencing, sequence shows that building a successful plasmid correctly, and confront a grain of extraction and purification, observe the virus infection expression in HEK293 cells after 24h, function and expression in detection of the virus.Second:By trypsin digestion method for isolation and culture of mouse embryonic fibroblasts, growth curve and cell cycle of MEF in vitro. The effects of the gestational age (days) Of mouse embryos and the time of digestion on the isolation and culture of MEF were assessed.Third:With empty virus infection MEF fluorescent protein packed, and found out the optimum value of MEF infection. Fourth:Will grow to be in good condition of MEF cells were divided into 3 groups at random:the purpose gene infection, empty virus infection group and normal control group, and carry four purpose gene adenovirus mixed infection of MEF, under inverted microscope observation period form change, finally it is concluded that the best experimental conditions.Results:First:After enzyme digestion identification of DNA electrophoresis that the vector was successfully constructed, gene sequencing is consistent with the target sequence, the titer of 1×1010PFU/ml. Second:The mouse embryo of 13.5 d is better than that of 10.5 d and 18.5d on the isolation of MEF. MEF in vitro is a kind of adherent cell With good ability for proliferation in passage 3. In room temperature, the digestive time of 0.25% trypsin should be 3-5 min. Third:Figure out the best interval of adenovirus infection MEF is 20-100, compared with normal group cell morphological changes, by long shuttle deformation for the half oval, with the empty viral infection group under the condition of not changing. Conclusion: the experiment successfully built a high drop degree of Oct4 and Sox2, C-Myc, Klf4 adenovirus vector, and the original generation of MEF cultivation, and infected mice fibroblast cells, observe the induction of MEF morphological changes, discusses the key problems of iPSC technology.