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Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies
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...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2011
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033823/ https://www.ncbi.nlm.nih.gov/pubmed/21244687 http://dx.doi.org/10.1186/1756-0500-4-8 |
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author | Park, Jae-Seung Surendran, Sneha Kamendulis, Lisa M Morral, Núria |
author_facet | Park, Jae-Seung Surendran, Sneha Kamendulis, Lisa M Morral, Núria |
author_sort | Park, Jae-Seung |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-3033823 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-30338232011-02-05 Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies Park, Jae-Seung Surendran, Sneha Kamendulis, Lisa M Morral, Núria BMC Res Notes Short Report 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. BioMed Central 2011-01-18 /pmc/articles/PMC3033823/ /pubmed/21244687 http://dx.doi.org/10.1186/1756-0500-4-8 Text en Copyright ©2011 Morral et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Short Report Park, Jae-Seung Surendran, Sneha Kamendulis, Lisa M Morral, Núria Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies |
title | Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies |
title_full | Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies |
title_fullStr | Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies |
title_full_unstemmed | Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies |
title_short | Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies |
title_sort | comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies |
topic | Short Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033823/ https://www.ncbi.nlm.nih.gov/pubmed/21244687 http://dx.doi.org/10.1186/1756-0500-4-8 |
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