The Construction And Transfection Of PcDNA3.1-hTERT Expression Vector

Mesenchymal stem cell (MSC), the un-haemopoietic stem cell which is isolated from bone marrow, can be differentiated into neuron-like cells under certain conditions. MSC contains not only characters of general stem cells but also more other fortes, for it is easily gotten, can be used for self-transplant, without the problems of ethics ,and easily to be transcripted ,and so on. It can provide seed cell for biological study ,and especially play an important role in tissue engineering. However, MSCs have a finite capacity to replicate in vitro and eventually enter a state of irreversible growth arrest for about 20 generations, and then become aging. At the same time, aging of cells is also a hot study spot in medicine, which blocks the development of human biology and tissue engineering, In recent years, some methods have been used to make the cells escape from senescence to immortalization, through virus, radioactive factors,oncogenes as well as proto-oncogene, and telomerase,etc. The most common way is by human telomerase reverse transcriptase (hTERT).Telomerase maintains cell viability and chromosomal stability through the addition of telomere repeats to chromosome ends. The reactivation of telomerase through the upregulation of TERT, the telomerase protein subunit, is an important step during cells immortalization. For that purpose, we transcriptased hTERT cDNA into human mesenchymal stem cells(hMSCs) by pcDNA3.1-hTERT expression vector, and used G418 to detect the integration,to lay a solid foundation for establishing an immortalized MSC line and even for basic and clinical research of neuroscience use.Methods:1. Cloning of hTERT cDNAThe total RNA was extracted from the tissue of esophageal carcinoma. Reverse transcript reaction and PCR were used to obtain the entire hTERT cDNA. The fragments of hTERT cDNA and pGEM-T Easy were ligated by T4 DNA ligase. After transferring them into the DH5a competent cells, the recombinant clones were selected and identified throng blue and white screening and PCR with the primers of T7/SP6.2. Constructing the eukaryotic expressing vector of hTERTInoculated the recombinant plasmid into liquid LB(Amp~+) culture medium and extracted the plasmid DNA. The DNA of pGEM-T-hTERT and pcDNA3.1 with restriction enzyme EcoR I were digested and purified respectively,then the fragements with sticky ends of hTERT gene and pcDNA3.1 were obtained. Ligated them with T4 DNA ligase and transformed them into the DH5a competent cells. Identified the positive clones with PCR.3. Transfecting of the recombinant eukaryotic expressing vector and detecting the integration of hTERTTransfected hMSC cells with pcDNA3.1- hTERT by Fugene 6 and detected the integration of hTERT byG418.Results:1. Cloning of hTERT cDNAThe full length of hTERT cDNA amplified by RT-PCR was 3363bp, which corresponded with its length provided by GenBank. After transformation of the ligated product as well as blue and white screening, many colonies were obtained. Through PCR with the primers T7/SP6, the positive transformed clones were identified. 2. Construction of the eukaryotic expressing vector of hTERTAfter PCR identification of the recombinant plasmid,using primers JD1 and JD2, a 642bp segment appeared clearly and brightly in agarose gel electrophoresis, which means the right insertion.3. Transfection of pcDNA3.1 -hTERT and identification of integratedAfter transfecting the the recombinant plasmid DNA vector into the hMSCs, positive cells clones were selected and cultured singly.G418 maked it clear that the ectopic hTERT gene had been stably integrated in hMSCs.Conclusions:hTERT cDNA was correctly cloned and the recombinant eukaryotic expression vector pcDNA3.1-hTERT was constructed successfully. The ectopic hTERT gene had been stably integrated in hMSCs. This research laid a solid foundation for establishing an immortalized MSC line and even for the basic and clinical research of neuroscience use.