title: Construction of an adenoviral expression vector carrying FLAG and hrGFP-1 genes and its expression in bone marrow mesenchymal stem cells.
abstract: The aim of this study was to construct an adenoviral expression vector for vascular endothelium growth factor 121 (VEGF121)-FLAG and humanized Renilla reniformis green fluorescent protein (hrGFP-1) genes, and to observe their expressions in bone marrow mesenchymal stem cells. Using pTG19T-VEGF121 as a template, polymerase chain reaction technology was adopted to mutate the VEGF121 gene by removing the stop codon and inserting NotI and XhoI restriction sites both before and after the gene sequences. The resultant gene was then subcloned into a pMD19-T plasmid, the pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids were double-digested, and small and large fragments were linked after gel recovery to complete the construction of recombinant adenovirus vectors. After titer determination, the recombinant adenovirus vectors were used to affect rabbit bone marrow mesenchymal stem cells, and fluorescence intensity was observed under fluorescence microscopy. Enzyme digestion identification and sequencing confirmed that the recombinant plasmids were successfully constructed, and observations under fluorescence microscopy showed significant expression of green fluorescent protein in recombinant adenovirus-infected bone marrow mesenchymal stem cells. The constructed adenoviral gene expression vectors carrying VEGF121-FLAG and hrGFP-1 can be expressed in eukaryotic cells, which may be used for gene therapy of ischemic disorders.
authors.affiliation: Department of Pharmacology, Liaoning Medical University, Jinzhou, Liaoning, China.
authors.forename: G X
authors.initials: GX
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, First People's Hospital of Jingmen, China.
authors.forename: L
authors.initials: L
authors.lastname: Hu
authors.affiliation: Department of Bone and Joint Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Bone and Joint Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou, China liudpcn@126.com.
authors.forename: D P
authors.initials: DP
authors.lastname: Liu
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009842:Oligopeptides; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009842:Oligopeptides; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins; C085706:FLAG peptide