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

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.