Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies.
Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies.
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DOI：
10.1186/1756-0500-4-8
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发表时间：
2011-01-18
影响因子：
1.8
通讯作者：
Morral, Nuria
中科院分区：
文献类型：
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作者：
Park, Jae-Seung;Surendran, Sneha;Morral, Nuria
背景技术背景：原代肝细胞是体外研究的最佳资源，这些研究旨在了解细胞和分子水平上的肝脏过程，这对于开发治疗非酒精性脂肪性肝炎、心血管疾病和2型糖尿病等高度流行疾病的新药是必要的。有必要确定简单的方法来遗传操纵原代肝细胞，并进行质粒，小干扰RNA（siRNA）或microRNA（miRNA）的功能研究。新的脂质体转染试剂是可用的，有可能产生更高水平的转染降低toxicity.FINDINGS：我们已经测试了几个基于脂质体的转染试剂用于分子生物学研究。我们表明，使用最近开发的制剂之一Metafectene Pro的转染效率对于质粒DNA（>45%的细胞）以及双链RNA（>90%的siRNA或microRNA）都很高。此外，所有这些核酸都存在可忽略的细胞毒性，即使细胞与DNA：脂质复合物孵育16小时。为了提供这些条件不仅可用于过表达感兴趣的基因，而且可用于RNA干扰应用的概念证明，我们使用质粒介导的短发夹RNA表达靶向两种肝脏表达的基因，固醇调节元件结合蛋白-1和脂肪酸结合蛋白5。此外，类似的转染条件被用来最佳地提供siRNA和microRNA.CONCLUSIONS：我们已经确定了一种脂质为基础的试剂，目前在分子生物学实验室中使用的核酸的原代肝细胞转染。这里描述的条件可用于加快各种各样的研究应用，从基因功能研究到microRNA靶标鉴定。
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.