[In vitro gene transfection into rabbit articular chondrocytes mediated by recombinant adeno-associated virus vector].
OBJECTIVE
To investigate the in vitro efficiency of recombinant adeno-associated virus (rAAV2) vector-mediated gene transfection into the rabbit articular chondrocytes.


METHODS
Articular chondrocytes were isolated from New Zealand rabbits, cultured, and transfected with rAAV2 containing enhanced green fluorescent protein (eGFP) gene at different values multiplicity of infection (MOI). Successful transfection of chondrocytes was confirmed by detection of recombinant enhanced green fluorescent protein using inverted fluorescence microscopy. The transfection efficiency was determined using the fluorescence-activated cell sorter. A maximum level of 4.76% was set as the background autofluorescence in live, uninfected chondrocytes.


RESULTS
The percentage of eGFP-positive chondrocytes increased in a vector dose-dependent manner. When the MOI value was <1 x 10(5) v.g./cell the transfection rate was positively linearly correlated with the dose. However, the MOI > 10(5) v.g./cell did not markedly increase the transfection rate. The greatest population of eGFP-expressing cells was 97.7% and the highest mean fluorescence intensity was 17.4 7 days after transfection. EGFP gene expression remained high until 56 days after transfection.


CONCLUSIONS
The in vitro efficiency of rAAV2 vector-mediated eGFP gene transfection into rabbit articular chondrocytes was high. The eGFP expression can be maintained for a rather long time. EGFP gene is an ideal tracer for chondrocytes transduction.
Li
Lian-hua
LH
Department of Orthopedics, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China.
Weng
Xi-sheng
XS
Qiu
Gui-xing
GX
Wu
Zhi-hong
ZH
Lin
Jin
J
Jin
Jin
J
Wang
Wei
W
Yang
Xin-yu
XY
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D000229:Dependovirus / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D011817:Rabbits; D011995:Recombination, Genetic; D014162:Transfection