[Preparation of the high-titer retroviral viruses for transducting PDGF-A gene].
OBJECTIVE
To prepare high-titer recombinant retrovirus for transducting platelet-derived growth factor A chain(PDGF-A) gene, which will pave the way for future research in vitro and in vivo on biological function of autocrine and paracrine of PDGF-A gene expression product.


METHODS
The cDNA fragment of human PDGF-A was inserted into the polyclonal sites of retroviral vector GINa containing cytomegalovirus (CMV) promotor. The constructed recombinant plasmid was transformed into PA317 packaging cell line. The positive clones were obtained by selection in G418-medium and amplified. The supernatant virus was used to infect NIH3T3 cells and titrated. After drug resistance selection, NIH3T3 cell clones were amplified, and a histochemical analysis was performed. SDS-PAGE electrophoresis and Western blot as well as 3H-TdR incorporation methods were adopted to assay the presence and the mitogenic activity of the PDGF in the conditioned medium.


RESULTS
The highest titer of the virus was 1.4 x 10(5) CFU/ml. The PDGF-A expression was verified with SDS-PAGE electrophoresis and Western blot and immunofluorescent staining. The cell mitogenic activitiy in the media was up to 51.2 U/(10(6).d).


CONCLUSIONS
We had made high-titer recombinant retrovirus for transducting human PDGF-A gene. The gene was expressed with high efficiency. The expressed product has significant mitogenic effect on NIH3T3 cell line.
Li
Y
Y
Department of Cardiology, Huashan Hospital, Shanghai Medical University, Shanghai 200040.
Wang
Z
Z
Kang
J
J
Chen
C
C
Dai
R
R
D003587:Cytomegalovirus / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D010957:Plasmids; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D018076:DNA, Complementary; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; C075278:platelet-derived growth factor A