Transfection of Lentivirus Vector Containing Apelin Gene into Umbilical Cord Mesenchymal Stem Cells in Vitro / 天津医药

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 infec-tion (MOI) to transfect human umbilical cord mesenchymal stem cells (hUCMSCs) and expression of target gene. Methods The apelin gene was chemically synthesized and amplified by polymerase chain reaction (PCR), and which was inserted into linear plasmid vector. The gene fragment and linear plasmid vector were connected by In-Fusion technology after enzyme di-gestion and transformed into competent DH5αcells. The positive clones of lentiviral expression vector were obtained after screening and followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and then the virus titers were determined. HUCMSCs were transfected with lentivirus vector in vitro via different values of MOI. The trans-fection efficiency was obtained according to the optimal MOI. The expression of target gene was detected by RT-PCR and Western blot assay. Results A 284 bp target gene fragment with the restriction sites was obtained by PCR and connected to the lentiviral vector. The positive clones of lentiviral expression vector were corresponded to the expected result. The lentivi-ral particles were successfully packaged. HUCMSCs could be transfected by the lentivirus vector with high efficiency. The mRNA and protein levels of target gene were stably up-regulated within 2 weeks. Conclusion The lentivirus vector pUbi-apelin-FLAG-pSV40-EGFP can transfect apelin gene into hUCMSCs with high efficiency. The infected cells can express high levels of apelin gene in two weeks.