Nucleofection induces non-specific changes in the metabolic activity of transfected cells

Abstract

Transfection has become an everyday technique widely used for functional studies in living cells. The choice of the particular transfection method is usually determined by its efficiency and toxicity, and possible functional consequences specific to the method used are normally overlooked. We describe here that nucleofection, a method increasingly used because of its convenience and high efficiency, increases the metabolic rate of some cancer cells, which can be misleading when used as a measure of proliferation. Moreover, nucleofection can alter the subcellular expression pattern of the transfected protein. These undesired effects are independent of the transfected nucleic acid, but depend on the particular cell line used. Therefore, the interpretation of functional data using this technology requires further controls and caution.

Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

“…This is usually well tolerated by target cells and not associated with voltage-induced cytotoxicity as still observed for electroporation-assisted delivery strategies to introduce CRISPR/Cas9 RNA and/or protein components. 28 , 81 , 82 The possibility to use novel pseudotyping glycoproteins makes it possible to confine delivery of our chimeric Gag.MS2 particles to specific cell types, as described for retroviral targeting strategies by Buchholz and co-workers. 83 , 84 , 85 Calculation of amounts of Cas9 (in either mRNA or protein form) and guide RNA administered for gene editing in recently published articles led to estimates in the range of 1–15 pg mRNA/cell (i.e., 4.2 × 10 5 –6.3 × 10 6 mRNA molecules/cell), 5–75 pg Cas9 protein/cell (i.e., 1.8 × 10 7 –2.8 × 10 8 protein molecules/cell), and 1–100 pg guide RNA/cell (i.e., 1.9 × 10 7 –1.9 × 10 10 guide RNA molecules/cell).…”

Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents

“…Nucleofection (a modified electroporation technology) is now one of the most effective non-viral methods for in vitro gene delivery. Electroporation-based nucleofection technique by Lonza allows high transfection efficiency in hard-to-transfect cells [ 41 – 43 ]. However, nucleofection relies on an expensive kit that varies from cell line to cell line.…”

“…Also, neither the content of the cell-dependent electroporation buffer nor the electroporation parameters are disclosed by the manufacturer. There are concerns that the unknown additives in the buffer used for nucleofection might affect the metabolic rate of some cancer cells and can alter the subcellular distribution of the recombinant protein in some cells [ 43 ]. Experimental comparison of sonoporation to lipofection and nucleofection in transfection applications of Jurkat and K562 non-adherent cell lines allowed us to demonstrate that sonoporation and nucleofection are equivalent in terms of cell viability and transfection efficiency.…”

Nucleofection of Expression Vectors Induces a Robust Interferon Response and Inhibition of Cell Proliferation

“…Anderson et al (2013) found that nucleofection induces transient Eif2a (eukaryotic initiation factor 2-a subunit) phosphorylation, which results in a general decrease in translation initiation events. Another group (Mello de Queiroz et al, 2012) reported a cell type-dependent increase in the metabolic rate of cells after nucleofection. Therefore, special consideration and additional controls must be taken into account when using transfection for studies of cell responses.…”