[Construction of recombinant plasmid pEGFP-BMP7 and its expression in rat bone marrow mesenchymal stem cells in vitro].
ABSTRACT To construct recombinant plasmid pEGFP-BMP7 and determine its expression in rat bone marrow mesenchymal stem cells (MSCs) in vitro.
cDNA of target gene was obtained from neonatal rat kidney by RT-PCR. After sequencing the target gene, the cDNA was subcloned into a eukaryote plasmid pEGFP-N1 by directed cloning and then digested with two restrictive endonucleases to verify the correctiveness of the recombinant plasmid pEGFP-BMP7. Rat bone marrow MSCs were transiently transfected with the pEGFP-BMP7 and transfection efficiency of the Green Fluorescent Protein (GFP) was determined. RT-PCR and immunocytochemical analysis were also performed to detect the expression of BMP7 in rat MSCs.
1 311 bp cDNA fragment was obtained by RT-PCR and sequence analysis showed it matched perfectly with that of rat BMP7 gene except a single nucleotide change at 756 bp from T to A. Digestion of the recombinant plasmid showed two 1.3 kb and 4.7 kb fragments and their size were same as those of BMP7 and pEGFP. This indicated that BMP7 cDNA was successfully subcloned into pEGFP. Transient transfection showed an efficiency of 33% at day 2 in rat MSCs. After transfection, transcription of BMP7 was detected in MSCs and expression of BMP7 protein was also verified.
Recombinant eukaryote plasmid pEGFP-BMP7 was successfully constructed and expressed in rat bone marrow MSCs. This procedure may provide a unique method for stimulation of callus formation in distraction osteogenesis and reconstruction of craniofacial bone defects.