High-efficiency nonviral transfection of primary chondrocytes.
The introduction of foreign DNA into mammalian cells is an essential investigative tool in molecular biology. Nonviral approaches to transfection offer the advantage of relatively simple vector design, production, and purification and, for tissue engineering applications, avoid many of the potential risks associated with virus-mediated transfection methods. Unfortunately, primary cells, and in particular chondrocytes, are notoriously refractory to conventional transfection approaches, and optimized transfection efficiencies in these cells are extremely low (1-1.5%). In this chapter, we present three protocols that have proved useful in transfecting primary chondrocytes at high efficiency (~70%). The first uses radiofrequency electroporation, a transfection method that frequently works extremely well in cell types that are difficult to transfect. It should be noted that electroporation is not limited to DNA but that essentially any molecule can be introduced into the cell using this approach. In addition to the primary protocol, we present two additional reliable, albeit less efficient backup protocols, the first using exponential decay electroporation and the second FuGENE 6 transfection.
Department of Orthopedics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA.
Jean F
JF
Welter
Luis A
LA
Solchaga
Matthew C
MC
Stewart
D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000737:chemistry / Q000378:metabolism*; D018274:Electroporation / Q000295:instrumentation / Q000379:methods; D008055:Lipids / Q000235:genetics; D014162:Transfection / Q000379:methods*
C411955:FuGene; D008055:Lipids