High-efficiency transfection of nucleic acids by the modified calcium phosphate precipitation method in chondrocytes

Abstract

Extracellular matrix (ECM)-rich cartilage-derived chondrocytes are difficult to transfect with DNA/RNA. We modified the classical calcium phosphate transfection method by detaching adherent chondrocytes with trypsin and resuspending in CaPo4–nucleic acid precipitate followed by readherence for 24 h. Due to the absence of ECM, chondrocytes could be transfected with 80% efficiency. Potent gene silencing with several antisense oligonucleotides and small interfering RNAs and strong promoter–luciferase activity could be achieved. This approach is applicable to any adherent or suspended cells and has utility in gene knockdown, ectopic overexpression, promoter regulation studies, and gene delivery in tissue engineering and gene therapy applications.