Abstract
Abstract Transfection is an enabling technique for cellular and molecular biologists to study expression and knockdown of gene expression. Over the past decade, many researchers have utilized liposomal systems to deliver nucleic acids; however, this method has many caveats including low transfection efficiency and high cellular toxicity in some cell types. To address these shortcomings, Mirus Bio has developed a novel non-liposomal technology called the TransIT-X2™ Dynamic Delivery System. This unique polymeric delivery system is able to effectively condense negatively charged nucleic acids (plasmid DNA and/or siRNA), enter cells and break out of the endosomal compartment, a critical barrier for transfection. The plasmid and siRNA can then be delivered to their respective functional locations, the nucleus and the cytoplasm. In this study, we demonstrate functional transfection of labeled plasmid DNA co-delivered with labeled siRNA illustrating the dual abilities of the TransIT-X2 Dynamic Delivery System. Additionally, extensive head-to-head comparisons with a popular liposomal reagent, Lipofectamine® 2000 were performed using a luciferase expression assay in 30 cell types; TransIT-X2 demonstrated superior performance in 20 cell types with 11 showing greater than 2-fold higher expression compared to Lipofectamine 2000. Transfection efficiency of green fluorescent protein (GFP) was measured using flow cytometry and visualized by microscopy; most cell types had greater than 50% GFP efficiency. Lastly, gene knockdown after delivery of siRNA or miRNA was assessed. Two endogenous genes, GAPDH and AHA1, were targeted for siRNA knockdown and mRNA levels were assessed by qRT-PCR. miRNA function was assessed by the decrease in the miR-1 target mRNA PTK9. The TransIT-X2 Dynamic Delivery System shows effective knockdown via siRNA or miRNA. The TransIT-X2 Dynamic Delivery System offers researchers exceptional broad-spectrum transfection of plasmid DNA and siRNA. This cutting edge, non-liposomal polymeric delivery method enhances endocytic release of the transfection complexes allowing researchers to obtain very high transfection efficiencies. Citation Format: James Ludtke, Anthony Lauer, Austin Storck, Nicholas Rossi, Karen Neder, Anatoly Pinchuk, Laura Juckem. A novel dynamic delivery system enabling high efficiency transfection of cells. [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 479. doi:10.1158/1538-7445.AM2014-479
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