Glucocorticoid regulation of mouse mammary tumor virus sequences in transgenic mice

Abstract

We have introduced a chimeric plasmid, (12)(13)(14)(15)(16).As a first step to dissecting any MTV sequences involved in the mammary gland specific expression, we microinjected into murine embryos a chimeric plasmid that contains the MTV long terminal repeat (LTR) ligated to the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene with the "TATA" box and transcription initiation site and derived mice that contain germ-line copies of this plasmid.The ligation of these MTV sequences to the TK gene confers increased transcription over a basal level when cells transfected with this construct are grown in the presence of dexamethasone (this report; refs. 17 and 18). We find that the TK gene is expressed in the lactating mammary gland as well as in the ovaries of females of one line of mice. In addition, glucocorticoid-inducible transcription of the TK gene is detected in the testes of two independently derived males containing pLTR2TK in their germ line. MATERIALS AND METHODSConstruction of pLTR2TK Plasmid. The pLTR2 plasmid is subcloned from a permuted plasmid, pMTV2, which is cloned from viral DNA from an MTV-infected rat hepatoma cell line (4). For construction of the pLTR2TK plasmid see Fig. 1. The pLTR2 plasmid was cut with EcoRI and combined with an EcoRI fragment [excised from a pFG5 (22) Pronuclear Injections and DNA Transfections. Supercoiled or HindIII-linearized pLTR2TK was microinjected into one pronucleus of fertilized one-cell eggs recovered from the oviducts of either CD-1 (Charles River Breeding Laboratories) or C57BL/6 (The Jackson Laboratory) females (see Results) that had mated with males of the same strain the previous night. Approximately 5 pl of DNA or 1000-5000 copies of the plasmid were microinjected. After overnight cultivation to the two-cell stage in Whitten's medium (24), the eggs were implanted into pseudopregnant CD-1 females.Plasmid pLTR2TK was transfected into LMTK-mouse cells (25) by calcium phosphate precipitation. Twenty-four hours after the addition ofthe DNA precipitate, the cells were selected in HAT medium (0.5 mM hypoxanthine/0.4 ,M aminopterin/16 ,M thymidine), TK-positive colonies were selected (-2-3 weeks after selection) and grown. Cultures derived from single colonies were grown both in the presence and absence of 1 ,uM dexamethasone for 20-24 hr, and RNA was isolated from these.Isolation of RNA and DNA. Mice were anesthetized by intraperitoneal injection of Nembutal, and a lobe of the liver was removed through a small ventral incision. After homogenization in phosphate-buffered saline in a tissue grinder fitted with a Teflon pestle, the tissue was pelleted at 1000 rpm in an International centrifuge and lysed in 10 mM Tris, pH 7.5/100 mM NaCl/0.5% NaDodSO4/1 mM EDTA containing Abbreviations: MTV, mouse mammary tumor virus; LTR, long terminal repeat; TK, thymidine kinase; HSV-1, herpes simplex virus type 1; kb, kilobase(s); GRE, glucocorticoid regulatory element(s).