Generation Of Mouse IPS Cell Lines From MEFs Through Forced Expression Of Oct4, Klf4, C-Myc And Sox2

Objectives:1.Master technique for generating mouse iPS cells by lentivirus-mediated delivery of foreign genes into somatic cells.2.Generating MEF-derived iPS cell lines,which will be potentially employed to investigate the regulatory mechanisms on controlling the fates of iPS cells and perform the directed differentiation researches.Methods:1.Lentiviral vector of pHAGE2-EF1α-STEMMCA and pHAGE2-EF1aFull-ZsGreen-Wi indetified by enzyme digestion and PCR1) Identification of pHAGE2-EF1α-STEMMCA and pHAGE2-EF1aFull-ZsGreen-W by enzyme digestion:pHAGE2- EF1α-STEMMCA was cut by EcoRⅠ,KpnⅠ,EcoRⅠ& XhoⅠ,respectively. pHAGE2-EF1aFull-ZsGreen-W was cut by EcoRⅠ,PvuⅡ,BamHⅠ& XhoⅠ,respectively.2) Identification of pHAGE2-EF1α-STEMMCA by PCR:primers p1/p2,p3/p4,p5/p6 and p7/p8(specific for Oct4,Klf4,c-Myc and Sox2, respectively) were employed to amplify Oct4,Klf4,c-Myc and Sox2 from pHAGE2-EF1α-STEMMCA,respectively.2.Production of lentivirus carrying EGFP gene or Oct4-Klf4 geneAccording to standard protocol from Invitrogen,lentivirus carring EGFP gene or Oct4-Klf4 gene were produced,followed by confirming the successful production of lentivirus harboring EGFP and Oct4-Klf4 gene,respectively.3.In vitro assay for EGFP expression at the cellular levelPrior to reprogram MEFs,MEFs were infected with lentiviruses harboring EGFP gene to verify whether the target cells could be efficiently infected by lentivirus made from this lentiviral system through EGFP assay under fluorescence microscopy.4.Reprogramming MEFs to iPS cells by lentivirus-mediated gene transfer of Oct4,Klf4,c-Myc and Sox2 MEFs carrying Pou5f1-EGFP gene were infected with the concentrated lentiviruses harboring Oct4-Klf4 gene.12h after infection,the infected medium was replaced with fresh mouse ES cell medium.Change the medium every day until the colonies become big enough to be picked up around day 20 after infection.5.Establishment and characterization of mouse iPS cell lines1) Assay for EGFP expression under fluorescence microscopy2) Observe iPS cell proliferation and calculate their population doubling time3) Detect alkaline phosphatase(AKP) activity after the 5th passages4) Total RNA was isolated,RT-PCR was used to carry out to detect the expression of ES marker genes5) EB-based differentiation of iPS cells6) Teratoma formation by transplanting iPS cells into SCID miceResults:1.pHAGE2-EF1α-STEMMCA and pHAGE2-EF1aFull-ZsGreen-W indetified by enzyme digestion and PCR Lentiviral vectors were confirmed to be right by enzyme digestion and PCR2.Infecting efficiency of lentivirus and assay for EGFP expression MEFs could be efficiently infected by lentivirus made from this lentiviral system,and EGFP gene harbored by MEFs could normally express.3.Reprogramming MEFs to iPS cells by lentivirus-mediated gene transfer Colonies first become visible approximately in the 4th day after the lentiviral infection.They become large enough to be picked up around the 20th day after infection.4.Establishment and characterization of mouse iPS cell lines1) High level of EGFP expression iPS cell colonies could be detected in～20 days after infection2) 4 iPS cell lines had morphological characteristics of mES cells and alkaline phosphatase3) 4 iPS cell lines express ES cell-specific marker genes4) Cells from all iPS cell lines formed embryoid bodies(EBs) in vitro, and EBs could differentiate into myocardial cells 5) Cells from all iPS cell lines formed teratomas in vivoConclusion:1) Master technique for reprogramming MEFs to iPS cells by lentivirus- mediated gene transfer2) Mouse iPS cell lines are preliminarily generated.