A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response

The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are closely related orphan nuclear hormone receptors that play a critical role as xenobiotic sensors in mammals. Both receptors regulate the expression of genes involved in the biotransformation of chemicals in a ligand-dependent manner. As the ligand specificity of PXR and CAR have diverged between species, the prediction of in vivo PXR and CAR interactions with a drug are difficult to extrapolate from animals to humans. We report the development of what we believe are novel PXR- and CAR-humanized mice, generated using a knockin strategy, and Pxr- and Car-KO mice as well as a panel of mice including all possible combinations of these genetic alterations. The expression of human CAR and PXR was in the predicted tissues at physiological levels, and splice variants of both human receptors were expressed. The panel of mice will allow the dissection of the crosstalk between PXR and CAR in the response to different drugs. To demonstrate the utility of this panel of mice, we used the mice to show that the in vivo induction of Cyp3a11 and Cyp2b10 by phenobarbital was only mediated by CAR, although this compound is described as a PXR and CAR activator in vitro. This panel of mouse models is a useful tool to evaluate the roles of CAR and PXR in drug bioavailability, toxicity, and efficacy in humans

Reversible gene knockdown in mice using a tight, inducible shRNA expression system

RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance was generated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms

Efficiency of H1/U6-shRNA-mediated firefly luciferase (Fluc) knockdown in mice expressing the codon-optimized tetR

<p><b>Copyright information:</b></p><p>Taken from "Reversible gene knockdown in mice using a tight, inducible shRNA expression system"</p><p></p><p>Nucleic Acids Research 2007;35(7):e54-e54.</p><p>Published online 21 Mar 2007</p><p>PMCID:PMC1874634.</p><p>© 2007 The Author(s)</p> Each configuration (control, H1- shRNA, U6 shRNA) was analyzed using two to four mice at the age of 8–10 weeks, respectively. Percentages of shRNA-mediated repression of firefly luciferase activity with standard error of the mean are shown for untreated controls (gray bars) and in animals upon 10 days feeding with 2 mg/ml doxycycline in the drinking water (white bars). In negative control animals (black bars), the shRNA expression cassettes are removed through cre-mediated recombination. Relative values of relative firefly luciferase expression level in different organs were calculated by using luciferases activities for reference. () H1 promoter-driven shRNA expression. () shRNA expression through the U6 promoter

Schematic representation of the inducible system and gene targeting strategy

<p><b>Copyright information:</b></p><p>Taken from "Reversible gene knockdown in mice using a tight, inducible shRNA expression system"</p><p></p><p>Nucleic Acids Research 2007;35(7):e54-e54.</p><p>Published online 21 Mar 2007</p><p>PMCID:PMC1874634.</p><p>© 2007 The Author(s)</p> Principle of tetR-mediated control of shRNA expression. Transcription of the RNA polymerase III-dependent promoter is blocked in cells expressing the tet repressor (tetR). Upon induction by doxycycline, tetR is removed from the tet-operator sequences (tetO) inserted into the promoter, allowing transcription of shFluc. ShRNA expression leads to RNAi-mediated knockdown of the target gene firefly luciferase. Renilla luciferase is used for reference to quantify firefly luciferase activity. Scheme of the targeting strategy. ShRNA and reporter constructs were independently inserted into the locus by homologous recombination in ES cells. Genes encoding the (Rluc) and firefly luciferases (Fluc) along with an adenovirus splice acceptor sequence and a polyadenylation signal (pA) were placed downstream of the endogenous promoter. The Fluc-specific shRNA is expressed under the control of the U6-tet or H1-tet promoter, and terminated by five thymidines (shRNA). The -sites flanking the shRNA expression cassettes were used to generate a negative control through cre-mediated recombination in ES cells. Southern blot analysis of genomic DNA from transfected ES cell clones containing the shRNA- (lane #1 and #2) or the reporter-constructs (lanes #3 and #4). Homologous recombination at the locus is detectable by using EcoRV-digested genomic DNA and probe 1, resulting in a 11.7 kb band for the wt and a 2.5 kb band for targeted allele. E: EcoRV; X: XbaI; neo: FRT-flanked neomycin resistance gene; hyg: FRT-flanked hygromycin resistance gene. () Western blot analysis from protein extracts of ES cells expressing either the wt tetR or the itetR using tetR- or β-Actin-specific antiserum. Control: protein extracts from wt ES cells