Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies
1 Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut St, IB130, Indianapolis, Indiana 46202, USA
2 Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Dr., MS A-401, Indianapolis, Indiana 46202, USA
3 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS 4053, Indianapolis, Indiana 46202, USA
BMC Research Notes 2011, 4:8 doi:10.1186/1756-0500-4-8Published: 18 January 2011
Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA) or microRNA (miRNA). New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity.
We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells) as well as double stranded RNA (>90% with siRNA or microRNA). In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA.
We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.