Abstract
A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.
Highlights
PC12 cells are a cell line originating from pheochromocytoma in the rat adrenal medulla (Schaefer et al 1987) and grow in culture as undifferentiated neuroblasts
PC12 is an established rat pheochromocytoma cell line, which responds to exposure to nerve growth factor (NGF) with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons
One of the important features of PC12 cells is that they are small cells with a limited amount of cytoplasm and long doubling time. They possess remarkable ability to respond to nerve growth factor (NGF), a neurotrophic polypeptide, inducing morphological and biochemical changes resulting in differentiation of PC12 cells into a sympathetic neuron-like phenotype (Greene and Tischler 1976; Grau and Greene 2012; Nagase et al 2005; Dhar et al 2007; Park et al 2007)
Summary
PC12 cells are a cell line originating from pheochromocytoma in the rat adrenal medulla (Schaefer et al 1987) and grow in culture as undifferentiated neuroblasts. One of the important features of PC12 cells is that they are small cells with a limited amount of cytoplasm and long doubling time They possess remarkable ability to respond to nerve growth factor (NGF), a neurotrophic polypeptide, inducing morphological and biochemical changes resulting in differentiation of PC12 cells into a sympathetic neuron-like phenotype (Greene and Tischler 1976; Grau and Greene 2012; Nagase et al 2005; Dhar et al 2007; Park et al 2007). Handling and manipulation during transfection is a crucial step
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