Reproducible and inducible knockdown of gene expression in mice

Abstract

Summary: RNA interference (RNAi) has emerged as an efficient approach for rapid analysis of gene function. In mammalian cells, vector-based expression of small hairpin RNAs (shRNA) produces potent and stable gene knockdown effects. An inducible RNAi system with reproducible levels of siRNA expression will extend the usefulness of this methodology to the identification of gene functions within the developing or adult mouse. We present evidence that an RNA polymerase III-driven U6 promoter with stuffer sequences flanked by loxP sites inserted at three different sites within the promoter drives shRNA expression in a Cre recombinase-dependent manner. We utilized this approach to develop a generic strategy for the reproducible knockdown of gene expression in mice. By placing the inducible shRNA cassette into the ROSA26 locus of the mouse, we were able to generate reproducible levels of controlled expression of shRNA to produce discernable phenotypes in vitro and in vivo. This approach circumvents the prescreening of random integration in embryonic stem cell clones and further enables conditional gene knockdown with temporal and/or tissue specificity. This methodology should expedite large-scale functional studies.

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

“…Inducible RNAi raises the prospect of temporal and dosage-sensitive control over the induction of gene knockdown. Previously reported strategies for inducible RNAi in ES cells or transgenic mice include the use of tamoxifen-inducible Cre-recombinase to activate vector-based shRNAs and shRNAs integrated in a defined locus (10,11), as well as the use of a doxycyclineinducible shRNA cassette integrated in a defined locus (12,13). Here we describe an alternative drug-inducible system based on the Ainv15 ES cell line, in which an shRNA-mir cassette is directed to the constitutively active hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus upon Cre-mediated sitespecific recombination.…”

Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes

“…In the mouse, where homologous recombination can direct transgenes to any locus, the most popular site for targeted integration has been ROSA26, a locus found by protein trapping to drive ubiquitous expression of integrated transgenes 18 . However, expression studies and functional assays demonstrate that targeting transgenes to ROSA26 does not result in sufficiently high levels of transgene expression in every tissue 19 . Thus the differential activation of this well-characterized locus limits its usefulness to a subset of tissues.…”

Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation

“…Cotransfection of short hairpin (sh) RNA constructs against GFP and mouse Smo was used to knock down A1::Smo::GFP and endogenous Smo, respectively, in transfected cells ( Fig. S2) (35,36). Cells were serum starved to promote cilial assembly, then treated with ShhN ligand or mock medium.…”