title: Construction of eukaryotic expression plasmid human transforming growth factor beta3 and its transfection into precartilaginous stem cells.
abstract: 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.
authors.affiliation: Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Tie
authors.forename: You
authors.initials: Y
authors.lastname: Hong-bo
authors.forename: Guan
authors.initials: G
authors.lastname: Han-feng
authors.forename: Chen
authors.initials: C
authors.lastname: An-min
authors.forename: Li
authors.initials: L
authors.lastname: Feng
mesh_terms: 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*
chemical_list: D053782:Transforming Growth Factor beta3; D011094:Polyethyleneimine