Abstract:

[Abstract]   Objective  To construct a recombinant plasmid pUbi-apelin-FLAG-pSV40-EGFP and package with
lentivirus to co-express enhanced green fluorescent protein (EGFP) and apelin, and to investigate optimal multiple of infection (MOI) to transfect human umbilical cord mesenchymal stem cells (hUCMSCs) and expression of target gene.  Methods   he apelin gene was chemically synthesized and amplified by polymerase chain reaction (PCR), and which was inserted into inear plasmid vector. The gene fragment and linear plasmid vector were connected by In-Fusion technology after enzyme digestion and transformed into competent DH5αcells. The positive clones of lentiviral expression vector were obtained after creening and followed by sequencing. The lentiviral vector was used to transfect293T cells and package virus, and then the irus titers were determined. HUCMSCs were transfected with lentivirus vector in vitro via different values of MOI. The transfection efficiency was obtained according to the optimal MOI. The expression of target gene was detected by RT-PCR and estern blot assay.  Results   A284bp target gene fragment with the restriction sites was obtained by PCR and connected to he lentiviral vector. The positive clones of lentiviral expression vector were corresponded to the expected result. The lentiviral particles were successfully packaged. HUCMSCs could be transfected by the lentivirus vector with high efficiency. The RNA and protein levels of target gene were stably up-regulated within2weeks.  Conclusion   The lentivirus vector pUbiapelin-FLAG-pSV40-EGFP can transfect apelin gene into hUCMSCs with high efficiency. The infected cells can express igh levels of apelin gene in two weeks.

Key words: mesenchymal stem cell, Lentiviral vector, transfection, gene expression, apelin