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Expression of a fusion gene consisting of the mouse growth hormone-releasing hormone gene promoter linked to the SV40 T-antigen gene in transgenic mice
Molecular and Cellular Endocrinology 137 (1998) 161 – 168
Expression of a fusion gene consisting of the mouse growth hormone-releasing hormone gene promoter linked to the SV40 T-antigen gene in transgenic mice N. Nogues a, E. Magnan a, P. De Grandis a, M. Butz a, R.D. Kineman a, J.J. Kopchick b, L.A. Frohman a,* a
Department of Medicine, Uni6ersity of Illinois at Chicago, Department of Medicine (M/C 787), 840 South Wood Street, Chicago, IL 60612, USA b Edison Animal Biotechnology Center, Ohio Uni6ersity, Athens, Ohio 45701, USA Received 30 September 1997; accepted 16 December 1997
Abstract Limited information is available concerning the regulation of growth hormone-releasing hormone (GHRH) gene expression in the hypothalamus, largely because of the lack of a suitable cellular model. In an attempt to immortalize hypothalamic GHRH-producing neurons, we have generated a transgenic mouse model which expresses the simian virus 40 (SV40) T-antigen gene (Tag) under the control of the GHRH gene promoter. The transgene contains :5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene. This construct was microinjected into fertilized oocytes. Fourteen of 96 mice born had integrated the transgene. These mice were fertile and showed no signs of central or peripheral tumors. The pattern of expression of the SV40 Tag gene was analyzed in four different transgenic lines by RT-PCR. The tissues tested include: hypothalamus, pituitary, cortex, cerebellum, spinal cord, adrenal, testis, spleen and lung. Transgene expression was consistently detected in the hypothalamus of all lines. In addition, SV40 Tag expression was also detected in the hypothalamus by Northern blot analysis in two of the transgenic lines. SV40 Tag expression was also detected in the testis of all transgenic lines by RT-PCR. This result was not expected since the GHRH gene sequences present in the transgene do not include the testis-specific transcription initiation site previously described. This suggests that GHRH gene expression in the mouse testis can be directed by regulatory sequences located downstream of the testis specific transcription start site. We conclude that the promoter region of the GHRH gene included in this construct contains the regulatory elements necessary to drive hypothalamic and testis expression in vivo. In addition, all mice from one of the transgenic lines developed cataracts in both eyes. SV40 Tag expression was detected not only in eyes with cataracts, but also, to a lesser extent, in eyes from other transgenic lines. Furthermore, the endogenous GHRH gene was found to be expressed in the eyes of normal mice. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: GHRH; Hypothalamus; Transgenic; SV40 T-antigen; Testis; Eye
1. Introduction Progress in characterizing the regulation of growth hormone-releasing hormone (GHRH) gene expression has been limited by the lack of a suitable in vitro cell * Corresponding author. Tel.: +1 312 4131279; fax: + 1 312 4130342; e-mail: [email protected]
system. The hypothalamus contains only 2000–4000 GHRH producing neurons, primarily located within the arcuate nucleus (Sawchenko et al., 1985; Miki et al., 1996). This relatively small number of GHRH neurons and the absence of spontaneously occurring tumors that have been successfully propagated in culture have hindered the establishment of GHRH-producing cell lines. Targeting oncogene expression in transgenic mice
0303-7207/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 0 3 - 7 2 0 7 ( 9 7 ) 0 0 2 4 2 - 6
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Fig. 1. The GHRH-SV40 Tag fusion gene. The GHRH-SV40 Tag construct contains : 5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene. The 2.7 kb SV40 Tag fragment (striped box) contains the coding region for the large T and small t antigens, including the translation initiation codon and transcription termination sites. The untranslated GHRH gene sequences are indicated by black boxes and the GHRH coding region is indicated by white boxes.
to produce specific, immortalized cell lines which exhibit differentiated phenotypes has led to a considerable breakthrough in many systems, and the generation of useful cell lines where no naturally occurring tumors existed (Efrat et al., 1988; Paul et al., 1988; Nakamura et al., 1989; Bryce et al., 1993; Lew et al., 1993). Using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to target simian virus 40 (SV40) T-antigen gene (Tag) expression in transgenic mice, Mellon et al. successfully generated a differentiated neurosecretory clonal cell line that expressed the GnRH gene (Mellon et al., 1990). In an attempt to immortalize GHRH-producing hypothalamic neurons, we have employed a similar strategy and generated transgenic mice which express the SV40 Tag gene under the control of the GHRH gene promoter. In a previous study, we used 872 bp of the 5%-flanking region of the mouse GHRH gene to direct SV40 Tag expression but failed to detect hypothalamic-specific expression of the transgene (Pecori Giraldi et al., 1994). In contrast, all mice developed adrenal tumors of neuroectodermal origin which expressed the SV40 Tag gene. Therefore, in the present study we expanded the construct to include a larger fragment of the mouse GHRH gene promoter (5 kb), encompassing 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1. We now report that these sequences contain the regulatory elements necessary to drive hypothalamic as well as testis-specific expression, in vivo.
2. Materials and methods
2.1. Transgene construction An EcoRI genomic fragment containing : 3.5 kb of mouse GHRH gene 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, was excised from a cre recombined P1 clone (Genome Systems, St.
Louis, Missouri) and inserted into pBluescript (-SK). The SV40 Tag gene (provided by Dr D. Hanahan, University of California, San Francisco) was subsequently introduced downstream of the GHRH gene sequences, as a 2.7 kb SalI–XhoI fragment (Fig. 1). The resulting GHRH-SV40 Tag fusion gene was excised from vector sequences by BamHI digestion, purified and used for microinjection.
2.2. Production and identification of transgenic mice Transgenic mice were produced by microinjecting the GHRH-SV40 Tag linearized DNA into the male pronucleus of fertilized eggs of C57BL/6J × SJL/J mice, as previously described (Wagner et al., 1981). Animals carrying the transgene were initially identified by Slotblot analysis of tail DNA using a 400 bp Tag probe. Transgenic mice of subsequent generations were identified by PCR analysis using the following SV40 Tag specific primers: sense (5%-CTCAGCCACAGGTCTGT3%) and antisense (5%-TTGCCCTTGGACAGGCT-3%). Mice were housed at the University of Illinois in the Chicago Biological Research Laboratory under controlled environmental conditions (12-h light, 12-h dark), with food and water ad libitum. Experiments were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals.
2.3. RNA extraction Animals were killed by decapitation and tissues were collected and frozen in liquid nitrogen. Founder mice were sacrificed at :1 year of age whereas mice from established transgenic lines were killed at 2–3 months of age. For the study of GHRH gene expression in the mouse eye, non-transgenic mice of the same strain were sacrificed (2 months old) and their eyes dissected as follows: the outer part of the globe was incised using a scalpel; the lens was removed with a forceps and the
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retina was then gently teased apart from the eye cup. Finally, the remaining posterior portion of the eye, including the choroid, sclera, optic nerve and harderian gland, was trimmed with a scissors. Each of the dissected parts from ten eyes were pooled for RNA extraction. Total RNA was isolated by a single step guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987) using Tri Reagent (Molecular Research Center, Cincinnati, OH). When used for RT-PCR analysis, the RNA was further purified by a second extraction with phenol:chloroform before precipitation, as previously described (Aleppo et al., 1997).
2.4. Northern blot analysis Total RNA isolated from a single hypothalamus or pituitary was electrophoresed on a denaturing agarose gel and transferred overnight to a Nytran membrane (Schleicher and Schuell, Keene, NH). The membrane was hybridized with a 400 bp SV40 Tag probe in 50% formamide at 42°C, and subsequently washed 2× with 1 × SSC/1% SDS at room temperature for 15 min., 1 × with 1× SSC/1% SDS at 65°C for 15 min. and once with 0.5× SSC/0.5% SDS at 65°C for 15 min. The specific hybridization bands were visualized by autoradiography.
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3. Results
3.1. GHRH-Tag transgenic mice A construct containing :5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene (Fig. 1) was used to generate transgenic mice. Fourteen out of 96 mice born had integrated the transgene. The number of copies integrated, as assessed by Slot-blot analysis, ranged from one–four. These mice were fertile and showed no signs of central or peripheral tumors by histological examination or by gross anatomical observation at the time of sacrifice. Four different transgenic lines were established and mice from each lineage were used to analyze the pattern of transgene expression.
3.2. Transgene expression in the hypothalamus
2.5. RT-PCR analysis
Total RNA from different tissues was extracted and analyzed by RT-PCR using SV40 Tag specific primers to determine the pattern of SV40 Tag gene expression in the transgenic mice. The tissues tested included: pituitary (PT), adrenal (AD), hypothalamus (HT), cortex (CX), cerebellum (CB), lung (LG), testis (TS) and spinal cord (SC). The results from four transgenic lines are shown in Fig. 2. Total RNA obtained from an SV40 Tag-expressing neuroepithelial cell line (AD69), previously established in our laboratory (Pecori Giraldi et al., 1994), was used as a positive control. Testis RNA
Total RNA (1 mg) from individual tissues was reverse transcribed with oligo(dT) as primer, using the Superscript Preamplification System for First Strand cDNA Synthesis (GIBCO-BRL). One-tenth (2 ml) of the RT reaction was used for PCR amplification with SV40 Tag specific primers (see production and identification of transgenic mice for primer sequences). The PCR reaction was carried out in a 50 ml final volume that included 1× PCR buffer (Perkin Elmer, Norwalk, CT), 1.5 mM MgCl2, 200 mM of each dATP, dCTP, dGTP and dTTP (Boehringer Mannheim, Indianapolis, IN), 0.25 mM of each primer and 1.25 U Amplitaq (Perkin Elmer). Thirty five cycles of amplification were carried out at 95°C for 1 min, 57°C for 1 min, and 72°C for 1 min. Non-reverse transcribed control samples were amplified in parallel to confirm the absence of genomic DNA contamination. Endogenous mouse GHRH mRNA amplification was carried out using the following primers: sense (5%-CAGGAGTGAAGGATGCT-3%), located within exon 2, and antisense (5%-AAGGTCAGAGCTGAAGC-3%), located within exon 5. In this amplification, the PCR conditions were the same as above except for the annealing temperature, which was changed to 52°C.
Fig. 2. Tissue distribution of SV40 Tag gene expression in the transgenic mice. Total RNA from different tissues was analyzed by RT-PCR using SV40 Tag specific primers, which amplify a 407 bp product. The tissues include: pituitary (PT), adrenal (AD), hypothalamus (HT), cortex (CX), cerebellum (CB), lung (LG), testis (TS) and spinal cord (SC). The results from four transgenic lines (T1–T4) are shown. Total RNA from an SV40 Tag-expressing neuroepithelial cell line (AD69) was used as a positive control. Testis RNA samples that were not reverse transcribed (RT −), were used as a control for genomic DNA contamination.
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Fig. 3. Northern blot analysis of SV40 Tag expression. Shown are total RNA from single hypothalami (H) and pituitaries (P) of different transgenic lines (A-D). Hypothalamic and pituitary total RNA from a wild type (WT) non-transgenic mouse was used as negative controls. Total RNA (10 mg) from both the AD69 cells and the original adrenal tumor were used as positive controls. The two spliced mRNAs for small t- and large T-antigen are 2.2 and 2.4 kb, respectively.
samples which had not been reverse transcribed, were also analyzed in parallel to control for genomic DNA contamination. The expected 407 bp PCR product was consistently detected in the hypothalamus of all lines examined. The identity of the amplified product was further confirmed by digestion with a restriction enzyme (PstI) which has a unique site within the SV40 Tag 407 bp fragment (data not shown). In addition, as shown in Fig. 3, transgene expression in the hypothalamus was also detected by Northern blot analysis in two transgenic lines. In this preliminary experiment, total RNA isolated from a single hypothalamus and pituitary of transgenic mice of different lines were tested. Total RNA (10 mg) from both the AD69 cells and the original adrenal tumor from which this cell line originates (Pecori Giraldi et al., 1994), were used as positive controls. The SV40 small t- and large T-antigen mRNAs (2.2 and 2.4 kb respectively) were detected in the hypothalamus of only two transgenic mice. No expression was observed in the pituitary of any lineage, in agreement with the RT-PCR data.
3.3. Transgene expression in the testis SV40 Tag expression was also detected by RT-PCR in the testis of all transgenic lines (Fig. 2). This result was not expected since the GHRH gene sequences present in the transgene do not include the testis-specific transcription initiation site previously described (Srivastava et al., 1995). However, the 407 bp PCR product detected in the reverse transcribed RNA samples is not the result of genomic DNA contamination, because control samples of testicular RNA which had not been reverse transcribed did not exhibit PCR amplification with the SV40 Tag specific primers in any transgenic line. The level of expression of the SV40 Tag gene within the testis was relatively low compared with that in the hypothalamus and could not be detected by Northern blot analysis (data not shown).
We determined whether the GHRH gene sequences included in the transgene would also be capable of driving expression of the SV40 Tag gene in the placenta, another GHRH-expressing tissue. Placentas were obtained from pregnant females at gestational day 15– 17, a time at which the highest levels of GHRH placental expression are observed in the mouse (Suhr et al., 1989). The genotype of the fetuses was determined by PCR analysis of fetal tail genomic DNA, using the SV40 Tag specific primers. Total RNA obtained from placentas corresponding to transgenic fetuses (+ ) was subsequently analyzed by RT-PCR (Fig. 4). Total RNA from the AD69 cells was used as a positive control while RNA samples from non-transgenic placentas (−) were used as a negative control. As shown in Fig. 4 (left), no PCR amplification product was observed in the placental samples using the SV40 Tag specific primers. In contrast, the same reverse transcribed placental RNA samples showed a specific 386 bp amplified product when mouse GHRH primers were used (right), indicating that the absence of SV40 Tag specific PCR amplification was not due to a problem with RNA extraction or reverse transcription. The same results were obtained in transgenic placentas from two different lines, indicating that other GHRH gene regulatory sequences not included in the transgene, are required for placental expression in vivo.
3.4. Transgene expression in the eye All mice from one transgenic line (T1) developed cataracts in both eyes. This phenotype appeared to be linked to the transgene, as none of the non-transgenic littermates displayed cataracts. To determine whether the cataracts were due to ectopic SV40 Tag expression, we analyzed RNA samples obtained from eyes by RTPCR using the SV40 Tag specific primers. The results revealed that expression of the SV40 Tag gene occurred not only in the eyes with cataracts (transgenic line T1), but also, to a lesser extent, in the eyes from other transgenic lines (data not shown). This observation led us to investigate whether the endogenous GHRH gene is normally expressed in the eye. We, therefore, analyzed total RNA from three normal mouse eyes by RT-PCR, using the mouse GHRH specific primers, and observed that the GHRH gene is consistently expressed (data not shown). This result was also confirmed by hybridization with a mouse GHRH specific probe, after transfer of the PCR product to a nylon membrane (Fig. 5). This result provides an explanation for the SV40 Tag expression in the eyes of the transgenic mice, and also expands the list of tissues in which expression of the GHRH gene has been detected (Boronat et al., 1994; Matsubara et al., 1995). To further characterize the specific site of GHRH gene expression within the mouse eye, we dissected a
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Fig. 4. Analysis of transgene expression in the placenta by RT-PCR. Placentas were obtained from pregnant females at gestational day 15–17. Left: total RNA obtained from placentas corresponding to transgenic fetuses ( +) was analyzed using SV40 Tag specific primers, which amplify a 407 bp product. Total RNA from the AD69 cells was used as a positive control while RNA samples corresponding to non-transgenic placentas (− ) were used as negative controls. Right: the same reverse transcribed placental RNA samples were used for PCR amplification using mouse GHRH primers. A specific 386 bp amplified product was detected in all samples.
total of ten eyes and pooled separately the lens, retina, and the remainder of the eye, consisting of the choroid, sclera, optic nerve and harderian gland. Total RNA was extracted from these samples and RT-PCR analysis was carried out using the GHRH specific primers. GHRH expression was not detected in the lens or the retina but was detected in the residual eye components (data not shown). The identity of the amplified product was subsequently confirmed by hybridization with a GHRH specific probe.
Fig. 5. GHRH expression in the eye. Southern blot of total RNA from normal mouse eye by RT-PCR, using mouse GHRH specific primers, subsequent transfer of the PCR product onto a nylon membrane, and hybridization with a mouse GHRH cDNA specific probe. Total RNA from hypothalamus was used as a positive control.
4. Discussion The results of the present study demonstrate that the promoter region of the GHRH gene included in the transgene contains the regulatory elements necessary to drive hypothalamic expression in vivo. Mice carrying this construct, which includes 3.5 kb of 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1 sequences, fused to the SV40 Tag gene, have consistently shown expression of the SV40 Tag gene in the hypothalamus. This is in contrast to previous studies in which hypothalamic expression of potential GHRH regulatory sequences in transgenic mice was not detected (Botteri et al., 1987; Pecori Giraldi et al., 1994; Nogues et al., 1997). Botteri et al. used different fragments of the human GHRH gene promoter to drive SV40 Tag gene expression in transgenic mice (Botteri et al., 1987). In that study, no animals expressed the SV40 Tag gene in the hypothalamus or developed hypothalamic tumors. Rather, they developed thymic hyperplasia, apparently as a result of ectopic SV40 Tag expression in the thymus. Recently, another transgenic mouse model, carrying a construct that consisted of rat GHRH gene promoter sequences fused to the CAT reporter gene, has been described (Nogues et al., 1997). In those animals, the transgene was specifically expressed in the placenta but not in the hypothalamus. Although CAT expression was observed in the brain, it was localized in glial cells. In addition to these attempts, our group previously generated transgenic mice using a construct containing 872 bp of the mouse GHRH gene 5%-flanking region fused to the SV40 Tag gene (Pecori Giraldi
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et al., 1994). All mice carrying that transgene developed adrenal medulla tumors of neuroectodermal origin which expressed the SV40 Tag gene. However, again there was no evidence of hypothalamic expression. Taken together, the failure to detect appropriate hypothalamic expression in these previous attempts may be attributed to (1) the use of relatively short fragments of the GHRH promoter lacking important regulatory element(s), and/or (2) the use of heterologous promoter sequences with possible interspecies incompatibilities. Although the transgenic animals of the present study expressed the SV40 Tag gene in the hypothalamus, none developed hypothalamic tumors, as confirmed by histological analysis of brain sections from different transgenic lines. Previously, a differentiated GnRH-expressing cell line has been established by specifically targeting the SV40 Tag gene in transgenic mice, using the regulatory domain of the GnRH gene (Mellon et al., 1990), indicating that hypothalamic neurons are capable of immortalization by SV40 Tag. In addition, GHRH neurons are capable of undergoing spontaneous transformation, resulting in GHRH-producing gangliocytomas within the human hypothalamus and pituitary (Asa et al., 1984). There are several possible explanations for the absence of tumor formation in this study. First, the level of expression of the SV40 Tag gene may not have been suffficient to transform the cells. Neoplastic transformation may require critical levels of SV40 T-antigen (Efrat and Hanahan, 1989), as not all tissues expressing this oncogene develop tumors (Reynolds et al., 1988; Baetscher et al., 1991). In this respect, it should be noted that all the transgenic founders had integrated only one – four copies, which could explain in part the low levels of SV40 Tag expression detected. In agreement with this, we have been unable to detect the SV40 Tag protein by either Western blot or immunocytochemistry (data not shown), suggesting that the levels of SV40 Tag expression are too low to be detected by these methods. Second, it is also possible that expression of the GHRH gene, and therefore of the GHRH-SV40 Tag fusion gene, does not occur until GHRH producing neurons are postmitotic and too differentiated to be immortalized by this technique. In addition to the hypothalamus, we have detected expression of the transgene in the testis of all transgenic lines. Expression of the GHRH gene in the mouse testis has previously been reported (Suhr et al., 1989). However, in the rat, where most of the studies on the testicular GHRH have been carried out, Srivastava et al. have characterized a GHRH testis-specific promoter and transcription start site, located 10.7 kb upstream of the hypothalamic transcription start site (Srivastava et al., 1995). The rat and the mouse GHRH genes are very similar and even a placental-specific transcription start site and untranslated leader sequence has been charac-
terized in both species and shown to be highly homologous (Gonzalez-Crespo and Boronat, 1991; Mizobuchi et al., 1991; Mayo et al., 1995). We, therefore, presumed that a homologous upstream testis-specific transcription start site, would also exist in the mouse GHRH gene. Since these upstream GHRH sequences were not included in the GHRH-SV40 Tag construct, expression of the transgene in the testis was not anticipated. However, the present results suggest that GHRH gene expression in mouse testis can be directed by regulatory sequences located downstream of this presumed testis-specific transcription start site. GHRH gene expression in the placenta, as in the testis, has been shown to use an alternative transcription start site located further upstream of the hypothalamic start site (Mizobuchi et al., 1991). However, unlike the transgenic testis, transgenic placentas did not exhibit expression of the transgene. As previously mentioned, transcription of the mouse GHRH gene in the placenta initiates : 10 kb upstream from the hypothalamic transcription start site and generates, by alternative RNA processing, a GHRH mRNA with a unique untranslated exon 1 (Mizobuchi et al., 1991; Mayo et al., 1995). Our results indicate that the GHRH gene sequences included in the transgene, which do not contain the placental-specific promoter, are not suffficient to drive placental expression in vivo, in agreement with the tissue-specific promoter usage previously described (Mizobuchi et al., 1991; Mayo et al., 1996). Low levels of transgene expression were also detected in the adrenals (transgenic line T3), and in the spinal cord (transgenic lines T1 and T4). The transgenic mouse model previously developed in our laboratory, in which the transgene only contained 872 bp of mouse GHRH gene 5%-flanking region, had already exhibited adrenal SV40 Tag expression (Pecori Giraldi et al., 1994). Thus, transgene expression in this tissue may actually reflect the ability of GHRH gene regulatory sequences, included in both constructs, to drive expression in the adrenals. In fact, previous reports have shown appreciable expression of GHRH in both adrenals (in humans by radioimmunoassay (Shibasaki et al., 1984) and in rat by RT-PCR (Matsubara et al., 1995)) and spinal cord (in rat by RT-PCR (Matsubara et al., 1995)), supporting the concept that transgene expression in these two tissues may be specific. Similarly, the SV40 Tag expression detected in the eye of the present transgenic mice appears specific, since we have also detected endogenous GHRH gene expression in the normal mouse eye. GHRH expression in the eye has not been previously described except for a report by Shibasaki et al. who showed moderate amounts of GHRH in human optic chiasm by radioimmunoassay (Shibasaki et al., 1984). Our results from the analysis of GHRH gene expression in the dissected mouse eye suggest that the specific site of GHRH expression
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within the eye is either the choroid, sclera, optic nerve or harderian gland. Further studies will be necessary to determine the physiological significance of GHRH expression in the eye. In conclusion, we have successfully targeted SV40 Tag expression to the hypothalamus of transgenic mice using a 3.5 kb 5%-flanking region of the mouse GHRH gene and have made novel observations concerning GHRH expression in the testis and eye. Although we have not yet succeeded in immortalizing GHRH-producing neurons, the hypothalamic-specific expression of the SV40 Tag gene in this transgenic model raises the possibility that, with appropriate manipulation, a GHRH-expressing immortalized cell line could be generated from these animals in the future.
Acknowledgements This work was supported by USPHS Grant DK30667 and the Bane Scholar Fund. N. Nogue´s is a postdoctoral fellow of the Direccio´ General d’Universitats i Recerca, Generalitat de Catalunya. J.J. Kopchick is supported, in part, by the State of Ohio’s Eminent Scholars Program, which includes a grant from Milton and Lawrence Goll. We thank Dr Muayyad Al-Ubaidi for helpful assistance and discussion regarding transgene expression in the eye.
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Shibasaki, T., Kiyosawa, Y., Masuda, A., Nakahara, M., Imaki, T., Wakabayashi, I., Demura, H., Shizume, K., Ling, N., 1984. Distribution of growth hormone-releasing hormone-like immunoreactivity in human tissue extracts. J. Clin. Endocrinol. Metab. 59, 263 – 268. Srivastava, C.H., Monts, B.S., Rothrock, J.K., Peredo, M.J., Pescovitz, O.H., 1995. Presence of a spermatogenic-specific promoter in the rat growth hormone releasing hormone gene. Endocrinology
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136, 1502 – 1508. Suhr, S.T., Rahal, J.O., Mayo, K.E., 1989. Mouse growth hormonereleasing hormone: Precursor structure and expression in brain and placenta. Mol. Endocrinol. 3, 1693 – 1700. Wagner, T.E., Hoppe, P.C., Jollick, J.D., Scholl, D.R., Hodinka, R.L., Gault, J.B., 1981. Microinjection of a rabbit b-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sci. USA 78, 6376-6380.
Expression of a fusion gene consisting of the mouse growth hormone-releasing hormone gene promoter linked to the SV40 T-antigen gene in transgenic mice N. Nogues a, E. Magnan a, P. De Grandis a, M. Butz a, R.D. Kineman a, J.J. Kopchick b, L.A. Frohman a,* a
Department of Medicine, Uni6ersity of Illinois at Chicago, Department of Medicine (M/C 787), 840 South Wood Street, Chicago, IL 60612, USA b Edison Animal Biotechnology Center, Ohio Uni6ersity, Athens, Ohio 45701, USA Received 30 September 1997; accepted 16 December 1997
Abstract Limited information is available concerning the regulation of growth hormone-releasing hormone (GHRH) gene expression in the hypothalamus, largely because of the lack of a suitable cellular model. In an attempt to immortalize hypothalamic GHRH-producing neurons, we have generated a transgenic mouse model which expresses the simian virus 40 (SV40) T-antigen gene (Tag) under the control of the GHRH gene promoter. The transgene contains :5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene. This construct was microinjected into fertilized oocytes. Fourteen of 96 mice born had integrated the transgene. These mice were fertile and showed no signs of central or peripheral tumors. The pattern of expression of the SV40 Tag gene was analyzed in four different transgenic lines by RT-PCR. The tissues tested include: hypothalamus, pituitary, cortex, cerebellum, spinal cord, adrenal, testis, spleen and lung. Transgene expression was consistently detected in the hypothalamus of all lines. In addition, SV40 Tag expression was also detected in the hypothalamus by Northern blot analysis in two of the transgenic lines. SV40 Tag expression was also detected in the testis of all transgenic lines by RT-PCR. This result was not expected since the GHRH gene sequences present in the transgene do not include the testis-specific transcription initiation site previously described. This suggests that GHRH gene expression in the mouse testis can be directed by regulatory sequences located downstream of the testis specific transcription start site. We conclude that the promoter region of the GHRH gene included in this construct contains the regulatory elements necessary to drive hypothalamic and testis expression in vivo. In addition, all mice from one of the transgenic lines developed cataracts in both eyes. SV40 Tag expression was detected not only in eyes with cataracts, but also, to a lesser extent, in eyes from other transgenic lines. Furthermore, the endogenous GHRH gene was found to be expressed in the eyes of normal mice. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: GHRH; Hypothalamus; Transgenic; SV40 T-antigen; Testis; Eye
1. Introduction Progress in characterizing the regulation of growth hormone-releasing hormone (GHRH) gene expression has been limited by the lack of a suitable in vitro cell * Corresponding author. Tel.: +1 312 4131279; fax: + 1 312 4130342; e-mail: [email protected]
system. The hypothalamus contains only 2000–4000 GHRH producing neurons, primarily located within the arcuate nucleus (Sawchenko et al., 1985; Miki et al., 1996). This relatively small number of GHRH neurons and the absence of spontaneously occurring tumors that have been successfully propagated in culture have hindered the establishment of GHRH-producing cell lines. Targeting oncogene expression in transgenic mice
0303-7207/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 0 3 - 7 2 0 7 ( 9 7 ) 0 0 2 4 2 - 6
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Fig. 1. The GHRH-SV40 Tag fusion gene. The GHRH-SV40 Tag construct contains : 5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene. The 2.7 kb SV40 Tag fragment (striped box) contains the coding region for the large T and small t antigens, including the translation initiation codon and transcription termination sites. The untranslated GHRH gene sequences are indicated by black boxes and the GHRH coding region is indicated by white boxes.
to produce specific, immortalized cell lines which exhibit differentiated phenotypes has led to a considerable breakthrough in many systems, and the generation of useful cell lines where no naturally occurring tumors existed (Efrat et al., 1988; Paul et al., 1988; Nakamura et al., 1989; Bryce et al., 1993; Lew et al., 1993). Using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to target simian virus 40 (SV40) T-antigen gene (Tag) expression in transgenic mice, Mellon et al. successfully generated a differentiated neurosecretory clonal cell line that expressed the GnRH gene (Mellon et al., 1990). In an attempt to immortalize GHRH-producing hypothalamic neurons, we have employed a similar strategy and generated transgenic mice which express the SV40 Tag gene under the control of the GHRH gene promoter. In a previous study, we used 872 bp of the 5%-flanking region of the mouse GHRH gene to direct SV40 Tag expression but failed to detect hypothalamic-specific expression of the transgene (Pecori Giraldi et al., 1994). In contrast, all mice developed adrenal tumors of neuroectodermal origin which expressed the SV40 Tag gene. Therefore, in the present study we expanded the construct to include a larger fragment of the mouse GHRH gene promoter (5 kb), encompassing 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1. We now report that these sequences contain the regulatory elements necessary to drive hypothalamic as well as testis-specific expression, in vivo.
2. Materials and methods
2.1. Transgene construction An EcoRI genomic fragment containing : 3.5 kb of mouse GHRH gene 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, was excised from a cre recombined P1 clone (Genome Systems, St.
Louis, Missouri) and inserted into pBluescript (-SK). The SV40 Tag gene (provided by Dr D. Hanahan, University of California, San Francisco) was subsequently introduced downstream of the GHRH gene sequences, as a 2.7 kb SalI–XhoI fragment (Fig. 1). The resulting GHRH-SV40 Tag fusion gene was excised from vector sequences by BamHI digestion, purified and used for microinjection.
2.2. Production and identification of transgenic mice Transgenic mice were produced by microinjecting the GHRH-SV40 Tag linearized DNA into the male pronucleus of fertilized eggs of C57BL/6J × SJL/J mice, as previously described (Wagner et al., 1981). Animals carrying the transgene were initially identified by Slotblot analysis of tail DNA using a 400 bp Tag probe. Transgenic mice of subsequent generations were identified by PCR analysis using the following SV40 Tag specific primers: sense (5%-CTCAGCCACAGGTCTGT3%) and antisense (5%-TTGCCCTTGGACAGGCT-3%). Mice were housed at the University of Illinois in the Chicago Biological Research Laboratory under controlled environmental conditions (12-h light, 12-h dark), with food and water ad libitum. Experiments were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals.
2.3. RNA extraction Animals were killed by decapitation and tissues were collected and frozen in liquid nitrogen. Founder mice were sacrificed at :1 year of age whereas mice from established transgenic lines were killed at 2–3 months of age. For the study of GHRH gene expression in the mouse eye, non-transgenic mice of the same strain were sacrificed (2 months old) and their eyes dissected as follows: the outer part of the globe was incised using a scalpel; the lens was removed with a forceps and the
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retina was then gently teased apart from the eye cup. Finally, the remaining posterior portion of the eye, including the choroid, sclera, optic nerve and harderian gland, was trimmed with a scissors. Each of the dissected parts from ten eyes were pooled for RNA extraction. Total RNA was isolated by a single step guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987) using Tri Reagent (Molecular Research Center, Cincinnati, OH). When used for RT-PCR analysis, the RNA was further purified by a second extraction with phenol:chloroform before precipitation, as previously described (Aleppo et al., 1997).
2.4. Northern blot analysis Total RNA isolated from a single hypothalamus or pituitary was electrophoresed on a denaturing agarose gel and transferred overnight to a Nytran membrane (Schleicher and Schuell, Keene, NH). The membrane was hybridized with a 400 bp SV40 Tag probe in 50% formamide at 42°C, and subsequently washed 2× with 1 × SSC/1% SDS at room temperature for 15 min., 1 × with 1× SSC/1% SDS at 65°C for 15 min. and once with 0.5× SSC/0.5% SDS at 65°C for 15 min. The specific hybridization bands were visualized by autoradiography.
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3. Results
3.1. GHRH-Tag transgenic mice A construct containing :5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene (Fig. 1) was used to generate transgenic mice. Fourteen out of 96 mice born had integrated the transgene. The number of copies integrated, as assessed by Slot-blot analysis, ranged from one–four. These mice were fertile and showed no signs of central or peripheral tumors by histological examination or by gross anatomical observation at the time of sacrifice. Four different transgenic lines were established and mice from each lineage were used to analyze the pattern of transgene expression.
3.2. Transgene expression in the hypothalamus
2.5. RT-PCR analysis
Total RNA from different tissues was extracted and analyzed by RT-PCR using SV40 Tag specific primers to determine the pattern of SV40 Tag gene expression in the transgenic mice. The tissues tested included: pituitary (PT), adrenal (AD), hypothalamus (HT), cortex (CX), cerebellum (CB), lung (LG), testis (TS) and spinal cord (SC). The results from four transgenic lines are shown in Fig. 2. Total RNA obtained from an SV40 Tag-expressing neuroepithelial cell line (AD69), previously established in our laboratory (Pecori Giraldi et al., 1994), was used as a positive control. Testis RNA
Total RNA (1 mg) from individual tissues was reverse transcribed with oligo(dT) as primer, using the Superscript Preamplification System for First Strand cDNA Synthesis (GIBCO-BRL). One-tenth (2 ml) of the RT reaction was used for PCR amplification with SV40 Tag specific primers (see production and identification of transgenic mice for primer sequences). The PCR reaction was carried out in a 50 ml final volume that included 1× PCR buffer (Perkin Elmer, Norwalk, CT), 1.5 mM MgCl2, 200 mM of each dATP, dCTP, dGTP and dTTP (Boehringer Mannheim, Indianapolis, IN), 0.25 mM of each primer and 1.25 U Amplitaq (Perkin Elmer). Thirty five cycles of amplification were carried out at 95°C for 1 min, 57°C for 1 min, and 72°C for 1 min. Non-reverse transcribed control samples were amplified in parallel to confirm the absence of genomic DNA contamination. Endogenous mouse GHRH mRNA amplification was carried out using the following primers: sense (5%-CAGGAGTGAAGGATGCT-3%), located within exon 2, and antisense (5%-AAGGTCAGAGCTGAAGC-3%), located within exon 5. In this amplification, the PCR conditions were the same as above except for the annealing temperature, which was changed to 52°C.
Fig. 2. Tissue distribution of SV40 Tag gene expression in the transgenic mice. Total RNA from different tissues was analyzed by RT-PCR using SV40 Tag specific primers, which amplify a 407 bp product. The tissues include: pituitary (PT), adrenal (AD), hypothalamus (HT), cortex (CX), cerebellum (CB), lung (LG), testis (TS) and spinal cord (SC). The results from four transgenic lines (T1–T4) are shown. Total RNA from an SV40 Tag-expressing neuroepithelial cell line (AD69) was used as a positive control. Testis RNA samples that were not reverse transcribed (RT −), were used as a control for genomic DNA contamination.
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Fig. 3. Northern blot analysis of SV40 Tag expression. Shown are total RNA from single hypothalami (H) and pituitaries (P) of different transgenic lines (A-D). Hypothalamic and pituitary total RNA from a wild type (WT) non-transgenic mouse was used as negative controls. Total RNA (10 mg) from both the AD69 cells and the original adrenal tumor were used as positive controls. The two spliced mRNAs for small t- and large T-antigen are 2.2 and 2.4 kb, respectively.
samples which had not been reverse transcribed, were also analyzed in parallel to control for genomic DNA contamination. The expected 407 bp PCR product was consistently detected in the hypothalamus of all lines examined. The identity of the amplified product was further confirmed by digestion with a restriction enzyme (PstI) which has a unique site within the SV40 Tag 407 bp fragment (data not shown). In addition, as shown in Fig. 3, transgene expression in the hypothalamus was also detected by Northern blot analysis in two transgenic lines. In this preliminary experiment, total RNA isolated from a single hypothalamus and pituitary of transgenic mice of different lines were tested. Total RNA (10 mg) from both the AD69 cells and the original adrenal tumor from which this cell line originates (Pecori Giraldi et al., 1994), were used as positive controls. The SV40 small t- and large T-antigen mRNAs (2.2 and 2.4 kb respectively) were detected in the hypothalamus of only two transgenic mice. No expression was observed in the pituitary of any lineage, in agreement with the RT-PCR data.
3.3. Transgene expression in the testis SV40 Tag expression was also detected by RT-PCR in the testis of all transgenic lines (Fig. 2). This result was not expected since the GHRH gene sequences present in the transgene do not include the testis-specific transcription initiation site previously described (Srivastava et al., 1995). However, the 407 bp PCR product detected in the reverse transcribed RNA samples is not the result of genomic DNA contamination, because control samples of testicular RNA which had not been reverse transcribed did not exhibit PCR amplification with the SV40 Tag specific primers in any transgenic line. The level of expression of the SV40 Tag gene within the testis was relatively low compared with that in the hypothalamus and could not be detected by Northern blot analysis (data not shown).
We determined whether the GHRH gene sequences included in the transgene would also be capable of driving expression of the SV40 Tag gene in the placenta, another GHRH-expressing tissue. Placentas were obtained from pregnant females at gestational day 15– 17, a time at which the highest levels of GHRH placental expression are observed in the mouse (Suhr et al., 1989). The genotype of the fetuses was determined by PCR analysis of fetal tail genomic DNA, using the SV40 Tag specific primers. Total RNA obtained from placentas corresponding to transgenic fetuses (+ ) was subsequently analyzed by RT-PCR (Fig. 4). Total RNA from the AD69 cells was used as a positive control while RNA samples from non-transgenic placentas (−) were used as a negative control. As shown in Fig. 4 (left), no PCR amplification product was observed in the placental samples using the SV40 Tag specific primers. In contrast, the same reverse transcribed placental RNA samples showed a specific 386 bp amplified product when mouse GHRH primers were used (right), indicating that the absence of SV40 Tag specific PCR amplification was not due to a problem with RNA extraction or reverse transcription. The same results were obtained in transgenic placentas from two different lines, indicating that other GHRH gene regulatory sequences not included in the transgene, are required for placental expression in vivo.
3.4. Transgene expression in the eye All mice from one transgenic line (T1) developed cataracts in both eyes. This phenotype appeared to be linked to the transgene, as none of the non-transgenic littermates displayed cataracts. To determine whether the cataracts were due to ectopic SV40 Tag expression, we analyzed RNA samples obtained from eyes by RTPCR using the SV40 Tag specific primers. The results revealed that expression of the SV40 Tag gene occurred not only in the eyes with cataracts (transgenic line T1), but also, to a lesser extent, in the eyes from other transgenic lines (data not shown). This observation led us to investigate whether the endogenous GHRH gene is normally expressed in the eye. We, therefore, analyzed total RNA from three normal mouse eyes by RT-PCR, using the mouse GHRH specific primers, and observed that the GHRH gene is consistently expressed (data not shown). This result was also confirmed by hybridization with a mouse GHRH specific probe, after transfer of the PCR product to a nylon membrane (Fig. 5). This result provides an explanation for the SV40 Tag expression in the eyes of the transgenic mice, and also expands the list of tissues in which expression of the GHRH gene has been detected (Boronat et al., 1994; Matsubara et al., 1995). To further characterize the specific site of GHRH gene expression within the mouse eye, we dissected a
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Fig. 4. Analysis of transgene expression in the placenta by RT-PCR. Placentas were obtained from pregnant females at gestational day 15–17. Left: total RNA obtained from placentas corresponding to transgenic fetuses ( +) was analyzed using SV40 Tag specific primers, which amplify a 407 bp product. Total RNA from the AD69 cells was used as a positive control while RNA samples corresponding to non-transgenic placentas (− ) were used as negative controls. Right: the same reverse transcribed placental RNA samples were used for PCR amplification using mouse GHRH primers. A specific 386 bp amplified product was detected in all samples.
total of ten eyes and pooled separately the lens, retina, and the remainder of the eye, consisting of the choroid, sclera, optic nerve and harderian gland. Total RNA was extracted from these samples and RT-PCR analysis was carried out using the GHRH specific primers. GHRH expression was not detected in the lens or the retina but was detected in the residual eye components (data not shown). The identity of the amplified product was subsequently confirmed by hybridization with a GHRH specific probe.
Fig. 5. GHRH expression in the eye. Southern blot of total RNA from normal mouse eye by RT-PCR, using mouse GHRH specific primers, subsequent transfer of the PCR product onto a nylon membrane, and hybridization with a mouse GHRH cDNA specific probe. Total RNA from hypothalamus was used as a positive control.
4. Discussion The results of the present study demonstrate that the promoter region of the GHRH gene included in the transgene contains the regulatory elements necessary to drive hypothalamic expression in vivo. Mice carrying this construct, which includes 3.5 kb of 5%-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1 sequences, fused to the SV40 Tag gene, have consistently shown expression of the SV40 Tag gene in the hypothalamus. This is in contrast to previous studies in which hypothalamic expression of potential GHRH regulatory sequences in transgenic mice was not detected (Botteri et al., 1987; Pecori Giraldi et al., 1994; Nogues et al., 1997). Botteri et al. used different fragments of the human GHRH gene promoter to drive SV40 Tag gene expression in transgenic mice (Botteri et al., 1987). In that study, no animals expressed the SV40 Tag gene in the hypothalamus or developed hypothalamic tumors. Rather, they developed thymic hyperplasia, apparently as a result of ectopic SV40 Tag expression in the thymus. Recently, another transgenic mouse model, carrying a construct that consisted of rat GHRH gene promoter sequences fused to the CAT reporter gene, has been described (Nogues et al., 1997). In those animals, the transgene was specifically expressed in the placenta but not in the hypothalamus. Although CAT expression was observed in the brain, it was localized in glial cells. In addition to these attempts, our group previously generated transgenic mice using a construct containing 872 bp of the mouse GHRH gene 5%-flanking region fused to the SV40 Tag gene (Pecori Giraldi
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et al., 1994). All mice carrying that transgene developed adrenal medulla tumors of neuroectodermal origin which expressed the SV40 Tag gene. However, again there was no evidence of hypothalamic expression. Taken together, the failure to detect appropriate hypothalamic expression in these previous attempts may be attributed to (1) the use of relatively short fragments of the GHRH promoter lacking important regulatory element(s), and/or (2) the use of heterologous promoter sequences with possible interspecies incompatibilities. Although the transgenic animals of the present study expressed the SV40 Tag gene in the hypothalamus, none developed hypothalamic tumors, as confirmed by histological analysis of brain sections from different transgenic lines. Previously, a differentiated GnRH-expressing cell line has been established by specifically targeting the SV40 Tag gene in transgenic mice, using the regulatory domain of the GnRH gene (Mellon et al., 1990), indicating that hypothalamic neurons are capable of immortalization by SV40 Tag. In addition, GHRH neurons are capable of undergoing spontaneous transformation, resulting in GHRH-producing gangliocytomas within the human hypothalamus and pituitary (Asa et al., 1984). There are several possible explanations for the absence of tumor formation in this study. First, the level of expression of the SV40 Tag gene may not have been suffficient to transform the cells. Neoplastic transformation may require critical levels of SV40 T-antigen (Efrat and Hanahan, 1989), as not all tissues expressing this oncogene develop tumors (Reynolds et al., 1988; Baetscher et al., 1991). In this respect, it should be noted that all the transgenic founders had integrated only one – four copies, which could explain in part the low levels of SV40 Tag expression detected. In agreement with this, we have been unable to detect the SV40 Tag protein by either Western blot or immunocytochemistry (data not shown), suggesting that the levels of SV40 Tag expression are too low to be detected by these methods. Second, it is also possible that expression of the GHRH gene, and therefore of the GHRH-SV40 Tag fusion gene, does not occur until GHRH producing neurons are postmitotic and too differentiated to be immortalized by this technique. In addition to the hypothalamus, we have detected expression of the transgene in the testis of all transgenic lines. Expression of the GHRH gene in the mouse testis has previously been reported (Suhr et al., 1989). However, in the rat, where most of the studies on the testicular GHRH have been carried out, Srivastava et al. have characterized a GHRH testis-specific promoter and transcription start site, located 10.7 kb upstream of the hypothalamic transcription start site (Srivastava et al., 1995). The rat and the mouse GHRH genes are very similar and even a placental-specific transcription start site and untranslated leader sequence has been charac-
terized in both species and shown to be highly homologous (Gonzalez-Crespo and Boronat, 1991; Mizobuchi et al., 1991; Mayo et al., 1995). We, therefore, presumed that a homologous upstream testis-specific transcription start site, would also exist in the mouse GHRH gene. Since these upstream GHRH sequences were not included in the GHRH-SV40 Tag construct, expression of the transgene in the testis was not anticipated. However, the present results suggest that GHRH gene expression in mouse testis can be directed by regulatory sequences located downstream of this presumed testis-specific transcription start site. GHRH gene expression in the placenta, as in the testis, has been shown to use an alternative transcription start site located further upstream of the hypothalamic start site (Mizobuchi et al., 1991). However, unlike the transgenic testis, transgenic placentas did not exhibit expression of the transgene. As previously mentioned, transcription of the mouse GHRH gene in the placenta initiates : 10 kb upstream from the hypothalamic transcription start site and generates, by alternative RNA processing, a GHRH mRNA with a unique untranslated exon 1 (Mizobuchi et al., 1991; Mayo et al., 1995). Our results indicate that the GHRH gene sequences included in the transgene, which do not contain the placental-specific promoter, are not suffficient to drive placental expression in vivo, in agreement with the tissue-specific promoter usage previously described (Mizobuchi et al., 1991; Mayo et al., 1996). Low levels of transgene expression were also detected in the adrenals (transgenic line T3), and in the spinal cord (transgenic lines T1 and T4). The transgenic mouse model previously developed in our laboratory, in which the transgene only contained 872 bp of mouse GHRH gene 5%-flanking region, had already exhibited adrenal SV40 Tag expression (Pecori Giraldi et al., 1994). Thus, transgene expression in this tissue may actually reflect the ability of GHRH gene regulatory sequences, included in both constructs, to drive expression in the adrenals. In fact, previous reports have shown appreciable expression of GHRH in both adrenals (in humans by radioimmunoassay (Shibasaki et al., 1984) and in rat by RT-PCR (Matsubara et al., 1995)) and spinal cord (in rat by RT-PCR (Matsubara et al., 1995)), supporting the concept that transgene expression in these two tissues may be specific. Similarly, the SV40 Tag expression detected in the eye of the present transgenic mice appears specific, since we have also detected endogenous GHRH gene expression in the normal mouse eye. GHRH expression in the eye has not been previously described except for a report by Shibasaki et al. who showed moderate amounts of GHRH in human optic chiasm by radioimmunoassay (Shibasaki et al., 1984). Our results from the analysis of GHRH gene expression in the dissected mouse eye suggest that the specific site of GHRH expression
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within the eye is either the choroid, sclera, optic nerve or harderian gland. Further studies will be necessary to determine the physiological significance of GHRH expression in the eye. In conclusion, we have successfully targeted SV40 Tag expression to the hypothalamus of transgenic mice using a 3.5 kb 5%-flanking region of the mouse GHRH gene and have made novel observations concerning GHRH expression in the testis and eye. Although we have not yet succeeded in immortalizing GHRH-producing neurons, the hypothalamic-specific expression of the SV40 Tag gene in this transgenic model raises the possibility that, with appropriate manipulation, a GHRH-expressing immortalized cell line could be generated from these animals in the future.
Acknowledgements This work was supported by USPHS Grant DK30667 and the Bane Scholar Fund. N. Nogue´s is a postdoctoral fellow of the Direccio´ General d’Universitats i Recerca, Generalitat de Catalunya. J.J. Kopchick is supported, in part, by the State of Ohio’s Eminent Scholars Program, which includes a grant from Milton and Lawrence Goll. We thank Dr Muayyad Al-Ubaidi for helpful assistance and discussion regarding transgene expression in the eye.
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