Construction of eukaryotic expression plasmid human transforming growth factor beta3 and its transfection into precartilaginous stem cells.
OBJECTIVE
To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factor beta3 vector (hTGF-beta3) and transfecting it into rat's precartilaginous stem cells (PSCs).


METHODS
Gene engineering technique was introduced to construct eukaryotic expression plasmid pcDNA3.1 (+)-hTGF-beta3. PSCs of rats were isolated and purified with method of immunomagnetic microbeads. Then PSCs were cotransfected with plasmid hTGF-beta3 and pcDNA3.1 (+)-enhanced green fluorescence protein (EGFP) by liner polyethyleneimine (PEI). And 48 hours later the transient expression of EGFP was observed under a fluorescence microscope, and the expression of hTGF-beta3 was detected with reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA).


RESULTS
The sequences of the recombinants were consistent with that from Genebank. Cotransfection of EGFP provided fast visual confirmation of successful transduction. The hTGF-beta3 mRNA and protein expression could be detected by RT-PCR and ELISA.


CONCLUSIONS
The recombinant plasmid is correctly constructed and successfully transfected into rat's PSCs, which is an important step to treat epiphyseal injury or other osteo-cartilage diseases with transgenic therapy.
Tie
Liu
L
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Hong-bo
You
Y
Han-feng
Guan
G
An-min
Chen
C
Feng
Li
L
D000818:Animals; D002356:Cartilage / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D010957:Plasmids; D011094:Polyethyleneimine; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D053782:Transforming Growth Factor beta3 / Q000235:genetics*
D053782:Transforming Growth Factor beta3; D011094:Polyethyleneimine