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