Primary cells are a valuable tool for researchers and are often preferred over transformed or immortalized cell lines since they are biologically more relevant and resemble the in vivo situation much closer. Unfortunately, efficient gene transfer in primary cells is still limited. Whereas viral strategies are time consuming and involve safety risks, nonviral methods are often inefficient for most primary cells. Nucleofection has been proven to overcome these limitations. Here, we describe the Nucleofection protocol for efficient transfection of human umbilical vein endothelial cells. Using a combination of a cell type-specific solution and electrical conditions, transfection efficiencies up to 90% can be achieved while survival rate is more than 70%.

Cell lines isolated from tumors are an important tool to study cancer in vitro. They can be used for drug development as well as for understanding the basic mechanisms underlying cancer. Transfection of cancer cell lines with different molecules like plasmid DNA, siRNA or mRNA is often an integral part for this kind of research. Lonza's 4D-Nucleofector™ System is a modular system for the efficient transfection of primary cells and cell lines with a variety of substrates including plasmid DNA and siRNA. The 4D-Nucleofector™ X Unit supports the Nucleofection™ in two different formats. The aluminum-free 20µl Nucleocuvette™ Strip allows the transfection of low cell numbers down to 2x104 cells per reaction. As 16 reactions can be performed in parallel it is well suited for optimizing Nucleofection™ Conditions for cells lacking a ready-to-use Optimized Protocol. For higher cell numbers of up to 2x107 cells per reaction the same Nucleofection™ Conditions can be applied in the 100μl single Nucleocuvette™ Vessel. For higher throughput needs the 96-well Shuttle™ Add-on can be connected to the 4D-Nucleofector™ System. With this add-on six 20µl Nucleocuvette™ Strips can be processed in parallel allowing for screening applications or accelerating the optimization of transfection parameters for many cell types. In this study we used the 4D-Nucleofector™ System in combination with the 96-well Shuttle™ Add-on for optimizing transfection conditions for multiple cancer cell lines. An exemplary optimization process is depicted for the human prostate carcinoma cell line DU 145 and for the human colorectal adenocarcinoma cell line COLO 205. Optimization steps included the selection of the appropriate Nucleofector™ Solution, Nucleofector™ Program and plasmid DNA concentration. Transfection parameters were thereby optimized for a variety of adherent and non-adherent human cancer cell lines resulting in transfection efficiencies of up to 99%; while maintaining high cell viability. Citation Format: Jenny Schroeder, Ludger Altrogge, Elke Lorbach, Srinivasan Kokatam, Sabine Schaepermeier, Meike Weigel, Gina Andretta-Beu, Stefanie Buesch, Tamara Grabeck, Alexandra Krumnow, Sonja Spicker, Sampada Kallol, Preeti Kapoor, Andrea Toell. Efficient transfection of cancer cell lines using the 4D-NucleofectorTM System. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2014-3485

RNAi-mediated gene knockdown is a powerful tool that is successfully applied to identify gene function and to elucidate biological pathways. RNAi experiments and large-scale siRNA screens require the efficient delivery of highly functional and specific nucleic acid substrates into the appropriate cell system. Cell types relevant for immunological research, such as primary T cells and several suspension cell lines, are poorly accessible using reagent based transfection approaches. Nucleofection® is an established method for the effective, non-viral transfection of nucleic acids into primary and hard-to-transfect cell types. We expanded the method to the 96-well format making high throughput applications such as siRNA-library screenings possible in cell types highly relevant for immunological research. We here present data showing the efficient RNAi-mediated gene knockdown in various cell lines and primary cells using 96-well nucleofection. For example, data about the knockdown of Fas-mediated apoptosis in Jurkat cells will be presented. These studies were expanded to PLK1, which is a key regulator of mitotic progression in mammalian cells. Knockdown of PLK1 resulted in impaired proliferation and increased apoptosis. Starting from these single gene analyses RNAi-library screens are set up for the identification of anti-proliferative and pro-apoptotic genes.