The recent clinical success of genetically modified T-cell therapies underscores the urgent need to accelerate fundamental studies and functional screening methods in T lymphocytes. However, a facile and cost-effective method for efficient genetic engineering of T-cells remains elusive. Current approaches often rely on viral transduction, which is labor-intensive and requires stringent biosafety measures. Plasmid-based electroporation presents an affordable alternative, but remains underexplored in T-cells. Moreover, the availability of prototypical T-cell lines is limited. Here, we address these challenges by focusing on two immortalized murine T-cell lines, HT-2 and CTLL-2, which recapitulate key characteristics of primary T-cells, including cytotoxicity and cytokine-dependent proliferation. Alongside the widely used Jurkat T-cell line, HT-2 and CTLL-2 were successfully transfected with single or multiple genes with high efficiencies by means of optimized electroporation in a cuvette-based system. Notably, optimization of plasmid constructs enabled the delivery of large gene-of-interest (GOI) cargos of up to 6.5 kilobase pairs, as well as stable integration of a GOI into the genome via the Sleeping Beauty transposon system. We also developed advanced methodologies for CRISPR/Cas9-mediated gene editing in immortalized T lymphocytes, achieving knockout efficiencies of up to 97 % and homology-directed repair (HDR)-based targeted knock-in efficiencies of up to 70 %. We believe this optimized plasmid-based electroporation approach will contribute to advances in basic research on lymphocyte biology, as well as providing a practical, cost-effective tool for preclinical studies of T-cell therapies.
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