Efficient, specific, developmentally appropriate cre-mediated recombination in anterior pituitary gonadotropes and thyrotropes

Abstract

Tissue-specific expression of cre recombinase is a well-established genetic tool to analyze gene function, and it is limited only by the efficiency and specificity of available cre mouse strains. Here, we report the generation of a transgenic line containing a cre cassette with codon usage optimized for mammalian cells (iCre) under the control of a mouse glycoprotein hormone α-subunit (αGSU) regulatory sequences in a bacterial artificial chromosome genomic clone. Initial analysis of this transgenic line, Tg(αGSU-iCre), with cre reporter strains reveals onset of cre activity in the differentiating cells of the developing anterior pituitary gland at embryonic day 12.5, with a pattern characteristic of endogenous αGSU. In adult mice, αGSU-iCre was active in the anterior lobe of the pituitary gland and in the cells that produce αGSU (gonadotropes and thyrotropes) with high penetrance. Little or no activity was observed in other tissues, including skeletal and cardiac muscle, brain, kidney, lungs, testis, ovary, and liver. This αGSU-iCre line is suitable for efficient, specific, and developmentally regulated deletion of floxed alleles in anterior pituitary gonadotropes and thyrotropes.

Keywords: BAC; Cga; alpha-GSU; chorionic gonadotropin alpha subunit; site specific recombination; transgenic mouse.

Figure 1. Structure of the BAC Tg(αGSU-iCre) transgene

The position of the αGSU gene within the RP24-329P6 BAC is indicated, together with Zfp292 (80kb) and a portion of the Mobkl2b gene (60kb/from 208kb). The exon - intron structure of the αGSU gene is shown. The first exon is non-coding (light blue) and 4 exons are coding (blue rectangles). The recombination targeted the second αGSU exon, inserting the iCre coding sequence at the ATG of the αGSU gene and using the αGSU gene polyadenylation sequence. The 4.6 kb promoter used for the previous αGSU-Cre transgenic mouse is indicated.

Figure 2

Expression of the BAC Tg(αGSU-iCre) transgene in the developing pituitary gland at e12.5. X-Gal stained αGSU-iCre; Rosa26LacZ double transgenic embryos reveal activity of the αGSU-iCre transgene during development. Panel A: Whole X-gal stained double transgenic embryo (e12.5). Panel B: The mandible was removed from the embryo in panel A to visualize the pituitary (arrow). Panel C: Sagittal cryosection of X-gal stained double transgenic embryo showing expression in the forming anterior lobe of the pituitary. Section is counterstained with neutral red. Magnification bar represents 100μm.

Figure 3

Tg(αGSU-iCre) transgene is active primarily in the pituitary gland. Analysis of cre activity in adult tissues was examined by X-Gal staining of αGSU-iCre;Rosa26LacZ double transgenic mice. Cryo sections were counterstained with neutral red. For the ovary, Tg(αGSU-iCre) mice were crossed with the double-fluorescent cre reporter mTmG line, that expresses membrane targeted tandem dimer tomato (mT) prior to cre mediated excision and membrane targeted green fluorescent protein (mG) after excision (Muzumdar et al. 2007). Ovary sections were counterstained with DAPI (blue). All magnification bars represent 100μm.

Figure 4

Expression of the BAC Tg(αGSU-iCre) transgene in the adult pituitary gland. αGSU-iCre; Rosa26-EYFP double transgenic mouse reveals a high level of the αGSU-iCre activity in the anterior lobe of the pituitary, with no expression in the intermediate and posterior lobe. Panel A: Pituitary cryo section of a αGSU-iCre; Rosa26-EYFP double transgenic mouse stained with EGFP antibody. Panel B: Double immunohistochemistry analysis with antibody against EGFP (green) and αGSU (red) in pituitary cryosections from αGSU-iCre; Rosa26-EYFP mice. Cell nuclei were stained with DAPI (blue). A, anterior lobe; I, intermediate lobe; P, posterior lobe.

Figure 5

Tg(αGSU-iCre) is specific for thyrotropes and gonadotropes. Adult pituitary cryo sections from mice doubly transgenic for Tg(αGSU-iCre) and Rosa26EYFP reporter strain were stained by double immunohistochemistry with antibodies against EYFP (green) and each pituitary hormone (red). EYFP expression co-localizes with LHβ, FSHβ and TSHβ in pituitaries from αGSU-iCre; Rosa26-EYFP double transgenic mice; and not with ACTH, PRL, and GH. Cell nuclei were stained with DAPI (blue).