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 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.
Kang
Jinjoo
J
Department of Surgery and Biochemistry & Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, 1450 Biggy St. NRT6501, M/C9601, Los Angeles, CA 90033, USA.
Ramu
Swapnika
S
Lee
Sunju
S
Aguilar
Berenice
B
Ganesan
Sathish Kumar
SK
Yoo
Jaehyuk
J
Kalra
Vijay K
VK
Koh
Chester J
CJ
Hong
Young-Kwon
YK
D002021:Buffers; D002470:Cell Survival; D018274:Electroporation / Q000379:methods*; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D010710:Phosphates / Q000737:chemistry; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
D002021:Buffers; D010710:Phosphates; D034741:RNA, Small Interfering; D012965:Sodium Chloride