大鼠肝细胞原代培养物的转染

Transfection of Primary Cultures of Rat Hepatocytes.

作者信息

Edwards Mina, Wong Siew Cheng, Chotpadiwetkul Roongsiri, Smirlis Despina, Phillips Ian R, Shephard Elizabeth A

机构信息

Department of Biochemistry and Molecular Biology, University College London, London, UK.

Institute of Molecular and Cell Biology, Proteos, Singapore.

出版信息

Methods Mol Biol. 2006;320:273-282. doi: 10.1385/1-59259-998-2:273.

DOI:10.1385/1-59259-998-2:273

PMID:27699672

Abstract

Five different transfection reagents-calcium phosphate, TransFast™ Transfection Reagent, Superfect™ Transfection Reagent, Effectene™ Transfection Reagent, and Tfx™-20-were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids. The efficiency of transfection of each reagent was monitored using the dual luciferase reporter gene assay system. Superfect Transfection Reagent, Effectene Transfection Reagent and Tfx-20 were the most effective for the transfection of primary hepatocytes and gave comparable transfection efficiencies. Calcium phosphate was found to be the least effective transfection reagent and gave the most variable transfection results. Tfx-20 gave the least variable transfection results when different hepatocyte preparations were compared.

摘要

比较了五种不同的转染试剂——磷酸钙、TransFast™ 转染试剂、Superfect™ 转染试剂、Effectene™ 转染试剂和 Tfx™-20——有效转染雄性大鼠原代肝细胞培养物的能力。通过胶原酶灌注法分离肝细胞，然后在转染前在基质胶包被的平板上培养24小时。用pGL3-Control或pGL3-Basic质粒转染细胞。使用双荧光素酶报告基因检测系统监测每种试剂的转染效率。Superfect转染试剂、Effectene转染试剂和Tfx-20对原代肝细胞转染最有效，且转染效率相当。发现磷酸钙是最无效的转染试剂，转染结果变化最大。当比较不同的肝细胞制剂时，Tfx-20的转染结果变化最小。