Molecular and Cellular Endocrinology lOl(1994) 227-236 ELSEVIER Gene transfer into zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Xenopus hepatocytes: transcriptional regulation by members of the nuclear receptor superfamily Dominique Marilley, Abderrahim Mahfoudi, Walter Wahli * Institut de Biologic Animale, Universite’ de Lausanne, Ba^timent de Biologic. CH-1015 Lausanne, Switzerland (Received 1 November 1993; accepted 7 January 1994) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR Abstract A procedure to culture Xenopus laeuis hepatocytes that allows the cells in primary culture to be subjected to gene transfer experiments has been developed. The cultured cells continue to present tissue-specific markers such as expression of the albumin gene or estrogen-controlled vitellogenin gene expression, which are both restricted to liver. Two efficient and reproducible gene transfer procedures have been adapted to the Xenopus hepatocytes, namely lipofection and calcium phosphate-mediated precipitation. The transcription of transfected reporter genes controlled by estrogen-, glucocorticoid- or peroxisome proliferator-response elements was stimulated by endogenous or co-transfected receptor in a ligand-dependent manner. Furthermore, the expression of a reporter gene under the control of the entire promoter of the vitellogenin Bl gene mimicked the expression of the chromosomal vitellogenin gene with respect to basal and estrogen-induced activity. Thus, this culture-trans- fection system will prove very useful to study the regulation of genes expressed in the liver under the control of various hormones or xenobiotics. Key words: (Xenopus laeuis hepatocytes); Primary culture; Gene transfer; Vitellogenin gene Bl; Steroid receptor) 1. Introduction A great advantage of using cells in primary cultures for the analysis of tissue-specific gene expression is that they maintain phenotypic functions, while perma- nent cell lines are often dedifferentiated. Primary cul- tures of rat hepatocytes, for example, have been devel- oped, which express numerous liver-specific genes that were generally not active in hepatocyte-derived cell lines (Jefferson et al., 1984; Isom et al., 1982; Pasco et al., 1988). Similarly, primary cultures of Xenopus lueuis hepatocytes also retain tissue-specific morphological and biochemical characteristics, e.g. bile canaliculi with microvilli, synthesis of albumin or expression of genes regulated by steroid hormones (Wangh and Knowland, 1975; Green and Tata, 1976; Wahli and Weber, 1977; Wahli et al., 1978; Wangh and Schneider, 1982; Kawa- hara et al., 1989). Until recently, however, primary hepatocyte cultures were of limited use for molecular studies. Such cul- * Corresponding author. Tel.: +4121/692-2492; Fax: + 4121/692- 2540. tures were rather refractory to the introduction of exogenous genetic material. Thus, genetic analyses have been largely limited to laborious approaches such as the use of recombinant retrovirus gene transfer (Ledley et al., 1987; Wolff et al., 1987; Wilson et al., 1988) or somatic cell hybridization (Szpirer et al., 1980). Re- cently, two independent studies have presented modifi- cations of the calcium phosphate-mediated precipita- tion procedure providing a simple and reproducible transfection approach for adult rat hepatocytes in pri- mary culture (Pasco and Fagan, 1989; Rippe et al., 1990). Furthermore, other transfection techniques, such as electroporation and cationic lipid-mediated trans- fection, have also been successfully applied to rat hep- atocytes in .primary culture (Yoon et al., 1990; Pa- quereau and Le Cam, 1992; Jarnagin et al., 1992). Our interest is to investigate the mechanisms re- sponsible for the activation of tissue-specific genes by nuclear hormone receptors, especially in Xenopus lae- uis liver (Wahli and Ryffel, 1985). The analysis of hormone-controlled gene expression in Xenopus hepa- tocytes in primary culture represents an essential com- plement to the studies carried out so far with non- hepatic cell lines or nuclear extracts issued from hep- 0303-7207/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0303-7207(93)00018-S