Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due...

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Bibliographic Details
Published in:Analytical biochemistry 2009-03, Vol.386 (2), p.251-255
Main Authors: Kang, Jinjoo, Ramu, Swapnika, Lee, Sunju, Aguilar, Berenice, Ganesan, Sathish Kumar, Yoo, Jaehyuk, Kalra, Vijay K., Koh, Chester J., Hong, Young-Kwon
Format: Article
Language:English
Subjects:
Buffers
Cell Survival
Electroporation
Electroporation - methods
Endothelial Cells - metabolism
Humans
Nucleofection
Phosphate-buffered saline
Phosphates - chemistry
Primary endothelial cells
RNA, Small Interfering - genetics
RNA, Small Interfering - metabolism
Sodium Chloride - chemistry
Transfection - methods
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Summary:Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2008.12.021