An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

Abstract

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.

Non-Viral Transfection of Human T Lymphocytes

“…Thus, for the first time, it is possible to chemically transfect Jurkat cells with both a transfection efficiency and a viability above 80%. Such results have only been obtained so far with nucleofection kits [35], which are associated with high costs and might not be suitable for large-scale experiments.…”

“…While considerably better, than anything l-PEI can produce, the low viability in particular is a concern, since any attempt to improve this value must be expected to dramatically reduce the transfection efficiency. For comparison, the Nucleofection technology has been reported to reach transfection efficiencies of >85% at 90% viability [35].…”

Research and development of the high-frequency square-wave pulse electroporation system

“…For instance, the pulse amplitude, rise-time and fall time of the bell-shaped voltage pulse are important and are recommended to be clearly specified (Cemazar et al 2018). There are also several studies performed, which suggests, that the square-wave and exponential decay pulses have higher efficiency in comparison with other type of pulses (Chicaybam et al 2017;Miklavcic et al 2010).…”

Novel Cell Adhesion/Migration Pathways Are Predictive Markers of HDAC Inhibitor Resistance in Cutaneous T Cell Lymphoma

“…Sanger sequencing confirmed successful cloning. 1 μg of each luciferase plasmid and 15 ng of Promega's pNL1·1·TK vector (#N1501) were conucleofected into 1 × 10 6 HUT78 cells with an Amaxa Nucleofector 2b system using the X-001 program and a homemade nucleofection buffer (SM1) as described [15]. Cells were incubated for 24 h in IMDM (Gibco) supplemented with 20% FBS and 1% penicillin-streptomycin, centrifuged at 200g for 10 min, and resuspended in 200 μL media.…”