[Preliminary study of primarily cultured C57 articular cartilage transfected with plasmid IDO-EGFP by lipofectamine].
OBJECTIVE
To determine the transfection efficiency and transient expression of pIDO-EGFP gene in primarily cultured C57 articular cartilage of mice, and to establish a transfection method of the primarily cultured articular cartilage in mice.


METHODS
Plasmid IDO-EGFP was amplified in Escherichia coli. The primarily cultured mouse chondrocytes which were initially obtained from articular cartilage were cultured in vitro and transfected with pIDO-EGFP by lipofectamine2000 reagent under optimized condition. Transfection process and transient expression were evaluated by fluorescent microscopy and laser scanning confocal microscopy (LSCM), and transfection efficiency was determined by flow cytometry.


RESULTS
There was obvious expression of EGFP at 24 h after transfection. The transfection efficiency of pIDO-EGFP into primarily cultured mouse chondrocytes reached 36.43% at 48 hours and the transfection did not affect the process of cell adherence.


CONCLUSIONS
IDO gene has been successfully transfected into primarily cultured chondrocytes by means of lipofectamine2000 reagent and the chondrocytes can survive in vitro. Satisfactory efficiency of transient transfection can be reached under optimized condition, which will provide a basis for gene introduction and modification of tissue engineered cartilage.
Duan
Xiao-Hong
XH
Department of Otolaryngology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China. duanxhon@163.com
He
Xian-Hui
XH
Cui
Peng-Cheng
PC
Wang
Xiao-Yan
XY
Wu
Ming-Ming
MM
Shi
Jian-Bo
JB
Xu
Geng
G
Jiang
Xun
X
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology; D005057:Eukaryotic Cells / Q000166:cytology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics*; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000235:genetics*; D008055:Lipids; D051379:Mice; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics*; D023822:Tissue Engineering; D014048:Tolonium Chloride; D014162:Transfection / Q000379:methods*
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D008055:Lipids; C086724:Lipofectamine; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014048:Tolonium Chloride