Chloramphenicol Acetyltransferase Fusion Gene in Transgenic Mice
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Proc. Nati. Acad. Sci. USA Vol. 87, pp. 6176-6180, August 1990 Genetics Tissue-specific expression and methylation of a thyroglobulin- chloramphenicol acetyltransferase fusion gene in transgenic mice (chloramphenicol acetyltransferase assay/DNA methylation/thyroglobufin gene/transcription control cAMP) C. LEDENT*, M. PARMENTIER*, AND G. VASSART*t *Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire; and tService de Gdndtique Mddicale, Universitd Libre de Bruxelles, Campus Erasme, 808 Route de Lennik, 1070 Brussels, Belgium Communicated by M. Van Montagu, May 9, 1990 (received for review January 4, 1990) ABSTRACT Fusion genes containing 1600 or 2000 base of genes to correspond to the regions involved in the tissue pairs of the bovine thyroglobulin gene 5' flanking region and specificity of gene expression (14). the chloramphenicol acetyltransferase (CAT) coding sequence With the double aim ofcompleting the study on the Tg gene were constructed and used to generate transgenic mice. Alto- promoter and of designing a thyroid-specific vector, we gether, 24 independent transgenic lines were obtained, and the constructed Tg-chloramphenicol acetyltransferase (CAT) fu- expression ofthe transgene was assayed by measuring the CAT sion genes and tested their expression in transgenic mice. We activity in different tissues. Depending on the transgenic lines, demonstrate here that 1600 bp of the Tg gene promoter are the fusion gene was either silent in all tissues or specifically able to target the expression of a reporter gene to the thyroid expressed in the thyroid. The level of expression was found to gland and that the DH site is not necessary for this tissue- be highly variable from one line to another and to be regulated specific expression. We also show that a Hpa II restriction by thyrotropin in a manner similar to the natural thyroglobulin site located in the DH region is unmethylated in a tissue- gene. The methylation status of the integrated DNA was tested specific manner. by digestion of DNA extracted from thyroid and other tissues with the isochizomers Msp I and Hpa H. It was found that one ofthe Hpa H sites was demethylated specifically in the thyroid. MATERIALS AND METHODS Constructions. Two constructs that had been used previ- The main interest oftransgenic mice is the ability to introduce ously for transfection studies in cell cultures (10) were used into the mouse genome recombinant genes composed of a in the present work. TgCAT17 and TgCAT18 consisted ofthe promoter that will direct the expression of the construct to receptor gene CAT placed downstream of the 5' flanking selected cell types, and a gene that will encode a specific region of the bovine Tg gene. The constructs TgCAT17 and protein. Ectopic production or overproduction of a protein TgCAT18 were excised from the vector, separated on aga- can give new insight into its function (1, 2). For the many rose gel, and purified by adsorption to glass powder (Gene- proteins ofunknown function that are being discovered every clean, BIO 101, La Jolla, CA). The sequencing was per- day by the recombinant DNA technology, it can possibly formed by the dideoxynucleotide chain-termination method provide an initial indication of their physiological role (3). (15) with a 380A automated sequencer (Applied Biosystems). simian virus 40 Production of Transgenic Mice. Transgenic mice were Nonspecific promoters, such as the early generated as described (16). Briefly, DNA was dissolved in region (4) and the promoter for the metallothionein gene (5), 5 mM Tris-HCI, pH 7.4/0.15 mM EDTA to a final concen- have been gradually replaced by tissue-specific promoters tration of 1.5 pug/ml. C57BL/6J x DBA/2J F1 females were that allow more targeted projects to be conducted (6, 7). superovulated and mated with fertile F1 males. Single-cell Therefore, the need for tissue-specific promoters is increas- embryos were harvested, pronuclei were microinjected with ing, since the number of presently characterized promoters 1-2 p1 of DNA solution, and embryos surviving microinjec- (in terms ofexpression in transgenic mice) is extremely small tion were reimplanted into pseudopregnant C57BL/6J X compared with the variety of cell types. DBA/2J F1 females. Screening of transgenic animals was We have been studying gene expression in the thyroid made by Southern blotting (17) of DNA extracted from tail gland for many years (8) and therefore were interested in biopsies (16) and hybridization with a 32P-labeled TgCAT17 designing a promoter that would allow the targeted expres- probe obtained by the random priming method (18). sion of a gene in the thyroid of transgenic mice. The most CAT Assays. Thyroid glands and other tissues were re- abundant thyroid-specific protein is thyroglobulin (Tg), the moved immediately after cervical dislocation, frozen in liquid large (660 kDa) thyroid hormone precursor. Its 8450-base- nitrogen, and stored at -80'C. They were further homoge- pair (bp) mRNA is encoded by a gene spreading over >200 nized in 4 volumes (with a minimum of 100 Al) of 20 mM kilobases (kb) in the bovine genome (9). The molecular Tris-HCl (pH 7.6) containing 128 mM KCI, 2 mM MgCl2, 1 mg mechanisms involved in the tissue-specific and hormone- ofbovine serum albumin per ml, 1 mM phenylmethylsulfonyl dependant expression of the Tg gene have been studied in fluoride, 1 ,tM leupeptin, 0.1 mg of bacitracin per ml, and 1.5 thyrocytes in primary culture (10) and in the FRTL-5 cell line mM 1,10-phenanthroline. After a 5-min centrifugation (Mi- (11, 12). A cAMP-responsive region was found in the prox- crofuge, full speed), the supernatants were heated at 650C for imal part of the promoter (10). Study of the DNase I sensi- 5 min and centrifuged again to remove denatured proteins. tivity of DNA extracted from the thyroid gland revealed the CAT activity was assayed essentially as described (19). presence of a tissue-speciflic DNase I-hypersensitive (DH) Homogenates were added to a buffered solution to give a final site located between -1600 and -2000 bp from the transcrip- reaction volume of 100 jul and final concentrations of 100 mM tional start (13). Such DH sites have been shown in a variety Tris HCl (pH 8), 2 puCi (1 ,Ci = 37 kBq) of [3H]chloram- The publication costs of this article were defrayed in part by page charge Abbreviations: CAT, chloramphenicol acetyltransferase; DH, payment. This article must therefore be hereby marked "advertisement" DNase I hypersensitive; T3, triiodothyronine; Tg, thyroglobulin; in accordance with 18 U.S.C. §1734 solely to indicate this fact. TSH, thyrotropin; PTU, 6-n-propyl-2-thiouracil. 6176 Downloaded by guest on September 23, 2021 Genetics: Ledent et al. Proc. Natl. Acad. Sci. USA 87 (1990) 6177 phenicol per ml, 250 ,uM butyryl-CoA, 1 mg of bovine serum HYP Tg promoter CAT gene SV40 albumin per ml, 1 mM phenylmethylsulfonyl fluoride, 1 ,uM TgCAT 17 leupeptin, 0.1 mg of bacitracin per ml, and 1.5 mM 1,10- -2000 bp - 3680 bp phenanthroline. Reactions were incubated for 5 hr at 370C t t and terminated by extraction with 200 pi of 2:1 (vol/vol) PstI HindIII EcoRI tetramethylpentadecane (TMPD)/xylenes; 180 ,gl of the or- I I ganic phase was assayed in a liquid scintillation counter. TgCAT 18 Duplicate samples were assayed. Standards containing 10, 5, -1600 bp I 3240 bp 1, 0.5, 0.1, 0.01, and 0 milliunit(s) of purified CAT (Pharma- cia) were included in each assay. The background level was 400-800 cpm, and the assay was linear between 0.01 milliunit I 400 bp (limit of detection) and 10 milliunits of purified CAT. FIG. 1. Structure of the Tg-CAT fusion genes. The restriction Hormonal Treatments. Triiodothyronine (T3)-treated mice sites used for digestion of the genomic DNA in the Southern blotting were injected twice a day (5 izg/100 g of body weight) for a procedure are indicated: EcoRI, HindIII, and Pst I. HYP represents period of8 days. Animals treated with 6-n-propyl-2-thiouracil the thyroid-specific DH site described in the bovine Tg gene pro- (PTU, Sigma) received 0.5 mg of PTU per ml of drinking moter (13). SV40 represents the transcription termination and poly- water during 28 days. Blood samples were obtained by adenylylation signals derived from the simian virus 40 early region. cardiac puncture under ether anesthesia. Thyroids were dissected out and processed for CAT assay. T3 was assayed one mouse containing the TgCAT17 transgene (line 193) and by standard radioimmunoassay (Amerlex-MT3 RIA kit, Am- one mouse containing the TgCAT18 transgene (line 972). ersham). The efficiency ofT3 and PTU treatments on thyroid Considering all experiments made on the different lines metabolism was controlled by measuring the pertechnetate containing the TgCAT17 or TgCAT18 transgenes, CAT ac- uptake. Control or treated animals received an intraperito- tivity was never detected above the background level in any neal injection of 20 ,Ci of sodium [9'Te]pertechnetate 30 organ tested except the thyroid. In the thyroid itself, the min before sacrifice. Blood, thyroid, and muscle samples expression level appeared to vary considerably from one were collected and assayed for radioactivity. transgenic line to another (Fig. 4). For the TgCAT17 trans- gene, nine independent transgenic lines were tested for expression: three lines showed no expression at all, and six RESULTS lines showed a thyroid-specific expression ranging from 0.01 Constructions. To test the tissue-specificity of the bovine milliunit to 2 milliunits of CAT activity per mg of tissue. For Tg gene promoter, transgenic mice were generated that the TgCAT18 transgene, 13 independent lines were tested: 5 harbor a chimeric gene composed of the 5' flanking region of lines were silent, and in the other 8 lines, the activity ranged the bovine Tg gene fused to sequences encoding the bacterial between 0.01 milliunit and 100 milliunits per mg of thyroid enzyme CAT as a reporter molecule.