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Abstract Cell lines as a immortalized cell materials play an important role in exploring gene function,producing transgenic animals and discovering new drugs. However, because of difficulties in immortalizing primary cells, until now only a few types of cells were reported to be immortalized. In order to overcome this problem, we aimed to construct a recombination lentivirus expressing human telomerase reverse transcriptase to improve the efficiency of immortalization so as to establish cell lines. So, we cloned ORF of hTERT gene from the retrovirus vector of pBABE-neo-hTERT and inserted into pLV-puro vector to construct pLV-puro-hTERT. After that, lentivirus particals were successfully packaged and used to infect pig preadipocytes.Then, positive cell clones were selected by puromycin. Finally, we examined the expression of telomerase reverse transcriptase by Real-time PCR, and evaluated the ability of proliferation and differetiation by DAPI and Oil red O staining. The results showed that porcine preadipocytes infected with pLV-puro-hTERT lentivirus displayed a 12-fold increase in telomerase mRNA level compared to control(P<0.01) with significant enhancement in cell proliferative activity, whilst no effects of lentivirus on porcine preadipocyte differentiation were observed. Taken together, these results indicate that the lentivirus vector can be used to promote primary cells proliferation, meanwhile it doesn't change primary cells' original biological characters, so it provides us a powerful tool to immortalize more primary cells in the future.
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Received: 07 December 2012
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