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. 2008 Jul;20(7):909-16.
doi: 10.1111/j.1365-2826.2008.01746.x. Epub 2008 Apr 28.

Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse

A Wolfe  1 , S Divall, S P Singh, A A Nikrodhanond, A T Baria, W W Le, G E Hoffman, S Radovick
Affiliations

Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse

A Wolfe et al. J Neuroendocrinol. 2008 Jul.

Abstract

Gonadotrophin-releasing hormone (GnRH) neurones located within the brain are the final neuroendocrine output regulating the reproductive hormone axis. Their small number and scattered distribution in the hypothalamus make them particularly difficult to study in vivo. The Cre/loxP system is a valuable tool to delete genes in specific cells and tissues. We report the production of two mouse lines that express the CRE bacteriophage recombinase in a GnRH-specific manner. The first line, the GnRH-CRE mouse, contains a transgene in which CRE is under the control of the murine GnRH promoter and targets CRE expression specifically to GnRH neurones in the hypothalamus. The second line, the GnRH-CRETeR mouse, uses the same murine GnRH promoter to target CRE expression to GnRH neurones, but is modified to be constitutively repressed by a tetracycline repressor (TetR) expressed from a downstream tetracycline repressor gene engineered within the transgene. GnRH neurone-specific CRE expression can therefore be induced by treatment with doxycycline which relieves repression by TetR. These GnRH-CRE and GnRH-CRETeR mice can be used to study the function of genes expressed specifically in GnRH neurones. The GnRH-CRETeR mouse can be used to study genes that may have distinct roles in reproductive physiology during the various developmental stages.

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Figures

Fig.4
Fig.4
Gonadotrophin-releasing hormone (GnRH) neurones exhibit CRE recombinase activity in doxycycline treated GnRH-CRETeR mice. (a) The construct used to produce the GnRH-CRETeR mouse is shown. A −3446 bp fragment of the mouse GnRH promoter was fused upstream of CRE recombinase as for the GnRH-CRE mouse. The proximal region of the GnRH promoter was modified to insert two Tet operators (TetO2). In series, the transgene also contains the CMV promoter regulating the Tet repressor gene. The rabbit β-globulin intron II is inserted between the promoter and the gene to enhance expression of the TetR gene. (b) Coronal sections from a bitransgenic GnRH-CRETeR/ROSALacZ mouse treated with Dox. Section includes the medial and lateral septum and the DBB. Tissue was treated with X-gal to visualise β-galactosidase activity and is seen as blue. GnRH neurones were stained as above and are brown. A population of X-gal stained neurones are seen in the lateral septum and do not express GnRH. Nine GnRH expressing neurones are seen in the medial septum and DBB, eight of which colocalised with X-gal staining. (c) GnRH neurones at the level of the organum vasculosum of the lamina terminalis/preoptic area at × 100 magnification. Ten GnRH immunostained neurones are depicted and nine co-stain blue indicating CRE recombinase activity. The boxed region is further displayed at × 250 magnification.
Fig. 1
Fig. 1
Targeting of CRE recombinase to the hypothalamus of transgenic mice. (a) The construct used to target CRE recombinase is illustrated. A −3446 bp fragment of the mouse gonadotrophin-releasing hormone (GnRH) promoter was fused upstream of CRE recombinase. CRE recombinase gene contains a Kozak initiation site, a nuclear localisation signal, an intron, derived from the SV40 t-antigen gene and a poly(A) signal. (b) Western blot analysis using an antibody specific for CRE protein in the indicated tissues from a GnRH-CRE mouse, founder line 35. Tissues shown are hypothalamus (hyp), cerebral cortex (ctx), cerebellum (cbl), liver (lvr), heart (hrt) and lung (lng).
Fig. 2
Fig. 2
CRE recombinase is expressed in gonadotrophin-releasing hormone (GnRH) neurones. Coronal section of a brain from a single 4-month-old male GnRH-CRE mouse (founder 35). Both sections are hypothalamic immediately caudal to the level of the organum vasculosum of the lamina terminalis. On the left is staining for Cre alone with DAB/Ni showing nuclear blue staining. On the right is an adjacent section stained for Cre (blue nuclear) and for GnRH (brown cytoplasmic). Both of these images are at × 100 magnification. Several neurones are boxed and shown in the inset at × 250 magnification. Scale bars = 100 μm. Cell counts from all brain sections from this mouse are also shown.
Fig. 3
Fig. 3
Gonadotrophin-releasing hormone (GnRH) neurones exhibit CRE recombinase activity. Coronal sections (× 100 magnification) from a bitransgenic GnRH-CRE/ROSALacZ mouse. Section includes the medial and lateral septum and the diagonal band of Broca (DBB). Tissue was treated with X-gal to visualise β-galactosidase activity, seen as blue. GnRH neurones were stained as above and are brown. A population of X-gal stained neurones are seen in the lateral septum and do not express GnRH. A group of GnRH expressing neurones are seen in the medial septum and DBB, all of which colocalise with X-gal staining. The boxed region is further shown at × 250 magnification.
Fig. 5
Fig. 5
CRE recombinase activity is induced by doxycycline in the gonadotrophin-releasing hormone (GnRH)-CRETeR mouse. Coronal sections from a bitransgenic GnRH-CRETeR/ROSALacZ mouse treated with Dox (left, +DOX) and untreated (right, -DOX). The upper sections are stained for β-galactosidase (blue/black) and the lower sections have been stained for GnRH (brown). Scale bars = 100 μm.
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