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.

DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner

“…pH 6.5). While complexing, cells were washed 2× in icecold PBS at 100 × g. Cells were resuspended in ice-cold 100 μl final volume Chikabuffer 1 52 and nucleofected in a 1D-nucleofector (program X-001). Immediately following nucleofection, cells were transferred to pre-warmed/equilibrated 10% serum RPMI for indicated time points.…”

3D-printing enabled micro-assembly of a microfluidic electroporation system for 3D tissue engineering

“…2d to g insets). This electric field condition is below literature reported, well-recognized electroporation field strength (~2000 V/cm for > 80% cell viability) for HeLa cells [37][38][39] . Meanwhile, our experimental study showed that our voltage condition at 400 V can achieve more than 90% cell viability (Fig.…”

CAR T cell therapy: A new era for cancer treatment (Review)

“…The Lonza Nucleofector II Electroporator performs effective genetic alteration of T cells using certain electric parameters and electroporation buffers (39). The electroporation of human T cells has been reported to be associated with ~40-60% of gene expression and 80% of cell viability (40). One of the limitations of electroporation is the low transfection efficiency and redundant cell damage (41).…”