Efficient, Specific, Developmentally Appropriate Cre-Mediated Recombination in Anterior Pituitary Gonadotropes and Thyrotropes

MARÍA INÉS PÉREZ MILLÁN; CAMPER SA; DAVIS SW

Houston, TX

Congreso; The Endocrine Society´s Annual Meeting; 2012

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 (1) under the control of a mouse glycoprotein hormone α-subunit (αGSU or Cga, chorionic gonadotropin alpha) regulatory sequences in a bacterial artificial chromosome (BAC) genomic clone. These transgenic mice produce efficient and specific cre-mediated recombination in gonadotrope and thyrotrope cells of the anterior pituitary gland with appropriate developmental onset. Pituitary deletion of transcription factors and signaling pathways was achieved with a smaller Cga transgene (2, 3, 4, 5). This transgenic line, Tg(Cga-cre)3Sac, has only 4.6 kb of mouse Cga promoter sequences, and its applicability was limited by ectopic activity in skeletal and cardiac muscle. To generate transgenic mice that more accurately recapitulate endogenous Cga gene expression we used homologous recombination in E. coli to introduce the improved cre cassette (icre) into a 228 kb mouse Cga genomic BAC clone in exon 1 at the translation start site. The engineered BAC was used to generate transgenic mice: Tg(Cga-icre). We used several different cre-reporter transgenic mice to assess the efficiency and specificity of the Tg(Cga-icre) BAC transgenic lines. A cre-activated lacZ reporter (R26R) revealed expression in the developing pituitary gland at embryonic day 12.5 in the appropriate pattern. Using the same reporter Tg(Cga-icre) activity was detected in the pituitary of 2 mo old adult mice, with little or no activity in inappropriate tissues such as muscle, brain, kidney, lungs, testis, ovaries, tail and liver. A cre-activated yellow fluorescent protein (YFP) reporter line revealed YFP co-localization with LHβ, FSHβ and TSHβ immunoreactive cells, but not with ACTH, PRL, and GH, indicating appropriate pituitary-cell specific restriction. In conclusion, we generated an improved Tg(Cga-icre) BAC transgenic line that targets pituitary thryrotropes and gonadotropes efficiently and specifically. This tool is an important addition to the armament of cre strains for targeting other pituitary cell types (6, 7, 8). BAC recombineered transgenes are an effective alternative to knock-in alleles that can create haploinsufficiency for the endogenous gene.