Construction of eukaryotic expression plasmid human transforming growth factor beta3 and its transfection into precartilaginous stem cells / 中华创伤杂志(英文版)
Chinese Journal of Traumatology
; (6): 288-293, 2007.
Article
in En
| WPRIM
| ID: wpr-236764
Responsible library:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Full text:
1
Index:
WPRIM
Main subject:
Plasmids
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Polyethyleneimine
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Stem Cells
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Enzyme-Linked Immunosorbent Assay
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Immunohistochemistry
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Transfection
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Cartilage
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Rats, Sprague-Dawley
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Reverse Transcriptase Polymerase Chain Reaction
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Cell Biology
Limits:
Animals
Language:
En
Journal:
Chinese Journal of Traumatology
Year:
2007
Type:
Article