[Construction of recombinant gene adenovirus encoding enhanced green fluorecence protein-peroxisome proliferator-activated receptor gamma2 fusion protein and its expression in bone marrow mesenchymal stem cells].
OBJECTIVE
To construct mouse enhanced green fluorecence protein (EGFP) -peroxisome proliferator-activated receptor (PPAR)gamma2, and to detect EGFP-PPARgamma2 expression in infected mouse bone marrow mesenchymal stem cells (BMSC).


METHODS
Cut the fragment of PPARgamma2 from the expression plasmid pcDNA flag PPARgamma2, then cloned the gene fragment into pEGFP-C1 and pEGFP-N1 vector. Subsequently, subclone the fragment EGFP-PPARgamma2 from pEGFP-C1-PPARgamma2 into the shuttle plasmid DC315. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid DC315-EGFP-PPARgamma2 and large adenovirus helper plasmid pBHGlox deltaE1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus Ad-EGFP-PPARgamma2 was confirmed. Then it was propagated in HEK293 cells. After the BMSC were transfected for 72 h, adipogenic differentiation was demonstrated.


RESULTS
HEK293 cells were transfected with the pEGFP-C1-PPARgamma2 or pEGFP-N1-PPARgamma2 in mediation of liposome. The former green fluorescence protein was better than the latter by fluorescence microscope. The recombinant plasmids were digested and identified. Western blot analysis showed the expression of EGFP-PPARgamma2 in vitro. EGFP-PPARgamma2 protein was detectable in the nucleus of BMSC.


CONCLUSIONS
The recombinant adenovirus encoding EGFP-PPARgamma2 fusion protein was successfully constructed, which provided a basis for application of EGFP-PPARgamma2 gene to adenovirus-mediated gene therapy.
Liao
Li-zi
LZ
Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, China.
Xiao
Jin-gang
JG
Yang
Miao-miao
MM
Kong
Zi-ren
ZR
Sun
Qin-ce
QC
Tian
Wei-dong
WD
D000256:Adenoviridae; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D051379:Mice; D047495:PPAR gamma / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D047495:PPAR gamma; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins