Construction and identification of recombinant lentivirus-mediated gene transfer system for rat transducer of regulated CREB activity 1*

ObjectiveTo construct a recombinant lentivirus vector which carries SD rat transducer of regulated CREB activity-1(TORC1) gene and examine its ability to express the TORC1 gene in vitro.MethodsThe coding sequence of SD rat TORC1 gene was amplified using PCR and cloned into pGC-FU vector. 293T cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles. When the cloned sequence was identified to be right, the recombinant lentivirus particles were amplified in a large quantity. The titer of virus was determined by real-time PCR and the level of TORC1 expression was examined by Western blot.ResultsThe recombinant lentivirus vector carrying TORC1 was constructed successfully and could express TORC1 at a high level in 293T cells in vitro, and the titer determined by real-time PCR was 2 × 108 TU/ml.ConclusionThe recombinant lentivirus vector could express TORC1 gene at a high level, and was very helpful in the study of exploring the effect of TORC1 on spinal cord injury.