Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues

Abstract

The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.

Methods for protein delivery into cells: from current approaches to future perspectives

“…This high input voltage leads to an increased temperature and pH changes in the area close to the electrodes [50,51]. Furthermore, the intense electric field causes lipid peroxidation and the generation of reactive oxygen species leading to the damage of proteins and DNA [52,53]. As a consequence, many cells die immediately after the procedure, whereas surviving cells have reduced viability and proliferative potential [54].…”

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening

“…Some cell types, such as HEK293T cells, lend themselves well to plasmid-based screening, since they can be transfected with efficiencies close to 100% using standard transfection reagents such as calcium phosphate, polyethylenimine, and lipid-based reagents. However, a number of other cell types including primary cells are refractory to these transfection methods; electroporation is typically used to overcome such problems ( Luft and Ketteler, 2015 ). It can be difficult to control the expression level of Cas9 and sgRNA by plasmid transfection, therefore this method may impose a trade-off between transfection efficiency and possible off-target activity and artifacts caused by overexpression of sgRNAs and Cas9.…”

Phenotypic screening with primary neurons to identify drug targets for regeneration and degeneration

“…Transfection is a gene manipulation process that occurs with the uptake of macromolecules such as plasmid DNA and RNA oligonucleotides (Luft and Ketteler, 2015). Transfection of neuronal cells using cDNA vectors is possible, but controlling expression levels is extremely difficult (Pearlberg et al, 2005; Moore et al, 2009).…”

“…Transfection of neuronal cells using cDNA vectors is possible, but controlling expression levels is extremely difficult (Pearlberg et al, 2005; Moore et al, 2009). Traditional transfection methods include lipid-based approaches and electroporation (Zeitelhofer et al, 2007), but both can be limiting by low efficiency, stressing or killing cells, and inducing an interferon response (Luft and Ketteler, 2015). …”