Ginot F, Decaux J F, Cognet M, Berbar T, Levrat F, Kahn A, Weber A
Unité de Recherches en Génétique et Pathologie Moléculaires, Unité 129 de l'Institut National de la Santé et de la Recherche Médicale, Paris, France.
Eur J Biochem. 1989 Mar 15;180(2):289-94. doi: 10.1111/j.1432-1033.1989.tb14646.x.
We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.
我们在本文中描述了一种在成年大鼠肝细胞原代培养物中研究瞬时基因表达的方法。通过胶原酶灌注分离后，单层培养的肝细胞在接种后的头24小时内通过磷酸钙沉淀技术用外源DNA进行转染。当用含有由猿猴病毒40早期启动子驱动的氯霉素乙酰转移酶基因的质粒转染时，肝细胞可重复性地高水平表达氯霉素乙酰转移酶（CAT）；这种瞬时表达远高于大鼠肝癌细胞系H4II所获得的表达水平。已经测试了不同的培养基条件；使用无血清、激素限定培养基可获得最佳水平的CAT活性。利用这些技术，我们研究了转入肝细胞的肝脏特异性基因的表达。我们表明，L-丙酮酸激酶启动子在这些肝细胞中具有活性，而在成纤维细胞中是沉默的。此外，使用无血清培养基可能有助于研究激素和营养物质在正常情况下对这些效应物有反应的细胞中的作用。
