Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

Jae Seung Park, Sneha Surendran, Lisa M. Kamendulis, Nuria Morral

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Background: 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. Findings. 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. Conclusions: 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.

Original languageEnglish
Article number8
JournalBMC Research Notes
Volume4
DOIs
StatePublished - 2011

Fingerprint

Gene Silencing
MicroRNAs
Nucleic Acids
Small Interfering RNA
Transfection
Hepatocytes
Genes
Molecular biology
Plasmids
Sterol Regulatory Element Binding Protein 1
Molecular Biology
Lipids
Fatty Acid-Binding Proteins
Double-Stranded RNA
DNA
Cytotoxicity
Medical problems
Liposomes
Liver
Toxicity

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies. / Park, Jae Seung; Surendran, Sneha; Kamendulis, Lisa M.; Morral, Nuria.

In: BMC Research Notes, Vol. 4, 8, 2011.

Research output: Contribution to journalArticle

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