Construction Of Tetracycline Regulated Gene Expression For Human Numb Gene In Two Recombinant Lentiviral Vectors

Objective:The purpose of this study was to construct tetracycline regulated gene exp ression for human Numb gene in two recombinant lentiviral vectors whose na mes were Lenti-EF1a-RTTA-IRES-Puro lentivirus and Lenti-EF1a-EGFP-TRE-Nu mb lentivirus.The Lenti-EF1a-RTTA-IRES-Puro lentivirus had the control compn ent for the expression of human Numb gene regulated by tetracycline,and the other one had the expressing compnent.We titered the lentiviral vectors titer aft er finish the construction.It would facilitate the following development of that human Numb gene influence the stemness of stem cells. Method:1.Enzyme digested the Lenti-egfp-IRES-puro plasmid to get the Lenti-IRES-puro frame.Rtta gene segment was verified by agarose gel electrophoresis(AGE).Rtta gene segment was linked to Lenti-IRES-puro frame to get Lenti-EF1a-RTT A-IRES-Puro recombinant plasmid.After amplification of recombinant plasmid i n competent Escherichia Coli,Plasmids was extrcted to verify by enzyme digest ion and sequencing technique.The Lenti-EF1a-RTTA-IRES-Puro recombinant pla smid was co-transfetion with packaging plasmids delta and p VSVG to 293 T ce lls.48 hours and 72 hours after transfection,we collected and concentrated the l entiviruse solution.Then lentiviruse solution was transfected to 293 T cells with an appropriately diluted concentration.48 hours later,titered the lentiviruse soluti on of Lenti-EF1a-EGFP-TRE-Numb by ELISA method.2.Lenti-EF1a-EGFP-TRE frame was get from Lenti-EF1a-EGFP-TRE-OCT4 plasmid by enzyme digestion.Human Numb gene segment was amplified from the total RNA of human embryonic stem cell by RT-PCR technique and verif ied by agarose gel electrophoresis.Then maked the Human Numb gene segment link to Lenti-EF1a-EGFP-TRE frame to get Lenti-EF1a-EGFP-TRE-Numb reco mbinant plasmid. After amplification of recombinant plasmid in competent Escherichia Coli,Plasmids was extrcted to verify by enzyme digestion and sequenci ng technique.The Lenti-EF1a-EGFP-TRE-Numb recombinant plasmid was co-tra nsfetion with packaging plasmids delta and pVSVG to 293 T cells.48 hours and72 hours after transfection,we collected and concentrated the lentiviruse soluti on.Then the lentiviruse solution was transfected to 293 T cells with an appropri ately diluted concentration.48 hours later,titered the lentiviruse solution of Lenti-EF1a-RTTA-IRES-Puro by DAPI stains way. Results:1.There were two genetic stripe in the picture of agarose gel electrophoresi s which were enzyme-digested product of Lenti-egfp-IRES-puro plasmid,that i ndicated it was successful to obtain the Lenti-IRES-puro frame.There was gene tic stripe in the picture of agarose gel electrophoresis at 1kbp nearby which is human Numb gene segment that indicated that it was successful to obtain the target gene segment.There were two genetic stripe in the picture of agarose ge l electrophoresis which were enzyme-digested product of Lenti-EF1a-RTTA-IRES-Puro recombinant plasmid.The sequencing result was 100% correct,that indica ted it was successful to obtain the Lenti-EF1a-RTTA-IRES-Puro recombinant pl asmid. The titer of the Lenti-EF1a-RTTA-IRES-Puro recombinant virus was 3.5*10^7TU/ml which were collected and concentrated at 48 hours after the co-t ransfection of the recombinant plasmid and ackaging plasmids to 293 T cells.An d the titer was 2.5*10^7TU/ml at the time of 72 hours.2.There was genetic stripe in the picture of agarose gel electrophoresis at 2kbp nearby who came from human total RNA by RT-PCR technique,that indica ted it was successful to obtain the human Numb gene segment.There were two genetic stripe in the picture of agarose gel electrophoresis which were enzymedigested product of Lenti-EF1a-EGFP-TRE-OCT4 plasmid,that indicated it was successful to obtain the Lenti-EF1a-EGFP-TRE frame.There were two genetic stripe in the picture of agarose gel electrophoresis which were enzyme-digested product of Lenti-EF1a-EGFP-TRE-Numb recombinant plasmid.The sequencing r esult was 100% correct,that indicated it was successful to obtain the Lenti-EF1a-EGFP-TRE-Numb recombinant plasmid. The titer of the Lenti-EF1a-EGFP-T RE-Numb recombinant virus was 6.5*10^7TU/ml which were collected and co ncentrated at 48 hours after the co-transfection of the recombinant plasmid and ackaging plasmids to 293 T cells.And the titer was 5.7*10^7TU/ml at the time of 72 hours. Conclusion:1. It was successful to obtain the Lenti-EF1a-RTTA-IRES-Puro recombinant lentivirus that had the control compnent for the expression of human Numb gene regulated by tetracycline.2.It was successful to obtain the Lenti-EF1a-EGFP-TRE-Numb recombinant lentivirus that had the expressing compnent for the expression of human Numb gene regulated by tetracycline.