Glucocorticoid Regulated Transcriptome in Wild-type and GR-C3 Knockin Mouse Embryonic Fibroblasts. Mus musculus

Glucocorticoids are primary stress hormones and their synthetic derivatives are widely used clinically. The therapeutic efficacy of these steroids is limited by severe side effects and glucocorticoid resistance. Multiple glucocorticoid receptor (GR) isoforms are produced by alternative translation initiation; however, the role individual isoforms play in controlling tissue-specific responses to glucocorticoids in vivo is unknown. We have generated knockin mice that exclusively express the most active receptor isoform, GR-C3. The GR-C3 knockin mice die at birth due to respiratory distress but can be completely rescued by antenatal glucocorticoid administration. To evaluate the GR-C3 transcriptome, we prepared mouse embryonic fibroblasts (MEFs) from E12.5 wild-type (WT) and GR-C3 knockin embryos and treated the cells with vehicle or the synthetic glucocorticoid Dexamethasone (Dex) for 6 hours. The GR-C3 isoform was found to have a markedly different gene-regulatory profile than GR in WT MEFs. Overall design: GR-C3 knockin mice were generated by standard gene targeting procedures. In brief, ES cells were generated in which exon 2 of the NR3C1 gene was replaced by a neomycin cassette. An exchange vector was generated containing a hygromycin cassette and a mutant NR3C1 exon 2 in which the ATG start codon for GR-C3 was left intact but mutated to ATC for each of the other seven translational isoforms. The exchange vector and Cre-recombinase were transfected into the targeted ES cells by electroporation, and positive ES cells were selected using G418 (sensitive) and hygromycin (resistant). A GR-C3 positive ES cell clone was injected into C57Bl/6 blastocysts to create chimeric mice. Chimeric males were bred with C57Bl/6 females for germline transmission, and the hygromycin cassette was deleted using Flp-deleter mice. The resulting mice were maintained on a C57Bl/6 background and intercrossed to produce WT, heterozygous GR-C3 knockin, and homozygous GR-C3 knockin mice. WT and homozygous GR-C3 knockin MEFs were generated from E12.5 embryos. WT and homozygous GR-C3 knockin MEFs cells were stimulated with either vehicle (control) or 100nM Dex for 6 hours. Total RNA was harvested for microarray analysis using the RNeasy Mini Kit (Qiagen) and RNase-Free DNase Kit (Qiagen) from 3 biological replicates of vehicle-treated WT MEFs, Dex-treated WT MEFs, vehicle-treated GR-C3 knockin MEFs, and Dex-treated GR-C3 knockin MEFs.