Construction Of Recombinant Lentiviral Expression Vector And Packaging Of The Lentiviral Vectors By Means Of Electroporation

Bown Marrow-derived stell cells(BMSCs) are used to cure many diseases because of its transversal differentiated poutency . But more and more studies show that BMSCs have powerful plasticity to contribute to tumorigenesis, therefore, it is necessary to do this research on safety of useing BMSCs.Many studies show that the formation of tumor cells highly depends on telomerase activity, so it is very important to obtain a high specificity and transcription activity human telomerase reverse transcriptase (hTERT) promotor for tumor gene therapy and controlling tumorigenesis during BMSCs transplant. In our study, we want to get a vector of lentivirus which contains an CD gene that could be promoted by our remoded hTERT .We cloned the CD gene from K12 genome by PCR, then cloned it into pLenti-hTERT-eGFP to get pLenti-hTERT-CD. In the next step, we got luciferase-ires fragment from plasmid Telve-C002 and cloned it into pLenti-hTERT-CD to get new constructed vectors called pLenti-hTERT-Luci-ires-CD. At last, we replaced the luciferase gene by eGFP to get the pLenti-hTERT-eGFP-ires-CD which we would package it into lentivirus .In order to get lentivirus quickly ,cheaply and high-effectively, we decided to package it by means of electroporation. Many studies have showed the transfection condition of electroporation, but we found molecular weight of the constructed vectors and the three package plasmids will also affect the package efficacy. So we tried to set up a new package system to get lentivirus in 293T cells high-effectively by electroporation. At last we detected titre of packaged lentivirus by FQ-PCR. The lentivirus titre which we packaged by electroporation was 106 TU/ml, it's enough for cells experiment.