Qureshi Hamid Yaqoob, Ahmad Rasheed, Zafarullah Muhammad
Department of Medicine, University of Montreal and Research Center of CHUM Notre Dame Hospital, K-5255 Mailloux, 1560 Sherbrooke Est., Montreal, Quebec, Canada H2L4M1.
Anal Biochem. 2008 Nov 15;382(2):138-40. doi: 10.1016/j.ab.2008.07.027. Epub 2008 Jul 31.
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.
富含细胞外基质（ECM）的软骨来源软骨细胞难以用DNA/RNA进行转染。我们对经典的磷酸钙转染方法进行了改进，先用胰蛋白酶分离贴壁软骨细胞，然后重悬于磷酸钙-核酸沉淀物中，再重新贴壁24小时。由于没有细胞外基质，软骨细胞的转染效率可达80%。使用几种反义寡核苷酸和小干扰RNA可实现有效的基因沉默，并具有很强的启动子-荧光素酶活性。这种方法适用于任何贴壁或悬浮细胞，在基因敲低、异位过表达、启动子调控研究以及组织工程和基因治疗应用中的基因递送方面都有用途。
