Abstract:

BACKGROUND: Bone marrow stromal cells (BMSCs) lack telomerase activity. Therefore, cell telomere length gradually shortens while proliferation in vitro, resulting in cellular senescence. This is an important factor that limits cell therapy application of BMSCs.
OBJECTIVE: To construct human telomerase catalytic subunit (hTERT) gene lentiviral expression vector, and to investigate the feasibility of transfection of human BMSCs with lentivirus-mediated hTERT gene.
METHODS: hTERT cDNA was obtained by PCR amplification from pReceiver-M02-hTERT. The coding sequence of hTERT was transferred into pDONR221 using BP recombination system, and then was transferred from pDONR221 into pLenti6.3/V5-DEST using LR clonase. The recombinant plasmid vector and packaging mixture were transfected into 293FT using Lipofectamine 2000 reagent, and the lentiviral particles were collected. ELISA was employed to detect the lentiviral titers. Q-PCR, Western blot and fluorescence immunocytochemistry were carried out to detect gene expression in human BMSCs after lentiviral transfecton.
RESULTS AND CONCLUSION: The hTERT gene lentiviral expression vector was constructed successfully. The average virus physical titer is 1.07×10¹² LP/L. Q-PCR confirmed that the hTERT gene expression level was significantly improved in modified human BMSCs. Western blot and immunocytochemistry confirmed the correct expression of hTERT protein in cells. The results suggest that transfection of human BMSCs with lentivirus-mediated hTERT gene can enhance hTERT expression.