An electroporation protocol for efficient DNA transfection in PC12 cells

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.

Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair

“…There are other more frequent methods to introduce foreign DNA into the nucleus of a cell, such as physical treatment (electroporation) [55], chemical carriers (cationic lipids) [56], or biological particles (viruses) as carriers [10]. These methods, especially for the viral carriers, have good transfection efficiency.…”

PKA activity exacerbates hypoxia-induced ROS formation and hypoxic injury in PC-12 cells

“…However, intracellular ATP levels were not significantly altered relative to empty vector controls and while a slight decrease in viability occurred after 24h SH in transfected 123.7 cells, it did not reach statistical significance. These discrepancies may result from the well-established and previously reported low transfectability of PC-12 cells [35, 36]. While we could express active PKA in 123.7 cells to a certain extent, all the cells may not have PKA activity.…”

“…Thus, the transfection may have only partially restored PKA cat-α activity in 123.7 cells or PKA may not have localized properly in the cell to affect ROS production and cell viability similarly to WT cells. Moreover, PC-12 cells do not transfect easily [35, 36] and there may be a significant number of cells with low or no PKA expression. Nevertheless the increase in ROS and slightly higher cell death in hypoxic transfected cells suggest that PKA plays a role in hypoxia-induced toxicity.…”

When 1 + 1 > 2: Nanostructured composites for hard tissue engineering applications

“…Various host-cell specific moieties and cell-penetrating peptides could be employed as functional surface groups with the aim to promote a more intimate tissue/material interface. As for the latter, enrichment of composites with analogues of lipofectamine, a positively charged compound used in gene transfection protocols to facilitate the diffusion of exogenous oligonucleotides across the cell membrane 201 , can be considered as an option. One of the peptides commonly used to improve the biocompatibility of the implant is Arg-Gly-Asp (RGD) 202,203 .…”