Establishment And Verification Of Human Embryonic Stem Cells With Over-expression Numb Gene

ObjectiveNumb is an important cell fate determination factor and plays an important role in determining the human embryonic stem cells division.In this study,the containing human Numb gene of lentiviral vector was used to infect human embryonic stem cells which provide experimental material for further study of Numb gene regulation in maintaining the properties of hESCs as stem cells.Method1.The Lenti-EF1a-RTTA-IRES-Puro recombinant lentivirus which was successful obtained in our lab was used to infect human embryonic stem cells.After p-uromycin screening,the human embryonic stem cell line which had the control component for the expression of Numb gene was established.Then use Lenti-EF1a-rtTA-IRES-EGFP recombinant lentivirus to infect the hESCs which was overexpress the rtTA gene to establish the complete tetracycline control system which was overexpress Numb gene in hESCs.2.The Lenti-EF1a-rtTA-IRES-EGFP recombinant lentivirus which was success-ful obtained in our lab was used to infect human embryonic stem cells to establish the cell line which overexpress EGFP gene.According to the EGFP gree fluorescent,pick out the Numb gene overexpression hESCs.Extract the DNA of clone to test the Numb integration to hESCs by RT-PCR.After different concentration of DOX inducing,Numb expression was test by Q-PCR.Results1.Successfully established people Numb gene expression of human embryonic stem cell lines,hair green fluorescent cloning was observed by fluorescence microscopy.2.Extract the DNA of clone to test the Numb integration to hESCs by RT-PCR.No.23 and 44 clone had obvious stripe in Maker Numb gene molecularsize.3.After DOX induce No.23 and 44 clone,the result of Q-PCR was that the expression of Numb was higher than control group(P<0.05).ConclusionIt was successful to establish and verificate human Numb gene overexpression human embryonic stem cells.