Novel and effective plasmid transfection protocols for functional analysis of genetic elements in human cardiac...

Cardiac fibroblasts, have lower gene transfer efficiency compared to dermal fibroblasts, posing challenges for plasmid-based gene transfer methods. A higher transfer efficiency could enable improved insight into heart pathology and development of novel therapeutic targets. In this study we compared eleven commercially available transfection reagents and eight plasmid purification methods. Finally, we systematically evaluated 150 unique transfection conditions (incubation times, addition of innate immune inhibitors, reagent to plasmid ratios etc) to optimize the methodology. The aim was to develop an optimized plasmid transfection protocol specifically tailored for primary human cardiac fibroblasts with high efficiency and minimal toxicity. While the actual transfection efficiency, indicated by the expression of fluorescent proteins, was less than 5%, our optimized protocol was sufficient for achieving significant gene expression levels needed for experimental applications such as luciferase enhancer-promoter assays. Leveraging our newly developed methodology, we could perform comprehensive profiling of nine viral and native enhancer/promoters, revealing regulatory sequences governing classical fibroblast marker (VIM) and resident cardiac fibroblast marker (TCF21) expression. We believe that these findings can help advance many aspects of cardiovascular research. In conclusion, we here report for the first time a plasmid transfection protocol for cardiac fibroblasts with minimal cell toxicity and sufficient efficiency for functional genomic studies.