High-efficiency transfection of nucleic acids by the modified calcium phosphate precipitation method in chondrocytes.
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 24h. 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.
Qureshi
Hamid Yaqoob
HY
Department of Medicine, University of Montreal and Research Center of CHUM Notre Dame Hospital, K-5255 Mailloux, 1560 Sherbrooke Est., Montreal, Quebec, Canada H2L4M1.
Ahmad
Rasheed
R
Zafarullah
Muhammad
M
D002130:Calcium Phosphates / Q000737:chemistry* / Q000378:metabolism; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D012313:RNA / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D012313:RNA; D004247:DNA; C020243:calcium phosphate