title: Phosphate-buffered saline-based nucleofection of primary endothelial cells.
abstract: 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.
authors.affiliation: 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.
authors.forename: Jinjoo
authors.initials: J
authors.lastname: Kang
authors.forename: Swapnika
authors.initials: S
authors.lastname: Ramu
authors.forename: Sunju
authors.initials: S
authors.lastname: Lee
authors.forename: Berenice
authors.initials: B
authors.lastname: Aguilar
authors.forename: Sathish Kumar
authors.initials: SK
authors.lastname: Ganesan
authors.forename: Jaehyuk
authors.initials: J
authors.lastname: Yoo
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Kalra
authors.forename: Chester J
authors.initials: CJ
authors.lastname: Koh
authors.forename: Young-Kwon
authors.initials: YK
authors.lastname: Hong
mesh_terms: 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*
chemical_list: D002021:Buffers; D010710:Phosphates; D034741:RNA, Small Interfering; D012965:Sodium Chloride