Construction of eukaryotic expression plasmid human transforming growth factor beta3 and its transfection into precartilaginous stem cells.
- Author:
Liu TIE
1
;
You HONG-BO
;
Guan HAN-FENG
;
Chen AN-MIN
;
Li FENG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cartilage; cytology; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Plasmids; Polyethyleneimine; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; metabolism; Transfection; methods; Transforming Growth Factor beta3; genetics
- From: Chinese Journal of Traumatology 2007;10(5):288-293
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo 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).
METHODSGene 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).
RESULTSThe 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.
CONCLUSIONSThe 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.
