Single copy shRNA configuration for ubiquitous gene knockdown in mice

Abstract

RNA interference through the expression of small hairpin RNA (shRNA) molecules has become a very promising tool in reverse mouse genetics as it may allow inexpensive and rapid gene function analysis in vivo. However, the prerequisites for ubiquitous and reproducible shRNA expression are not well defined. Here we show that a single copy shRNA-transgene can mediate body-wide gene silencing in mice when inserted in a defined locus of the genome. The most commonly used promoters for shRNA expression, H1 and U6, showed a comparably broad activity in this configuration. Taken together, the results define a novel approach for efficient interference with expression of defined genes in vivo. Moreover, we provide a rapid strategy for the production of gene knockdown mice combining recombinase mediated cassette exchange and tetraploid blastocyst complementation approaches.

Figure 1

(A) Scheme of the targeting strategy. ShRNA and reporter constructs were independently inserted into the rosa26 locus by homologous recombination in ES cells. Genes encoding the Renilla (Rluc) and firefly luciferases (Fluc) along with an adenovirus splice acceptor sequence and a polyadenylation signal (pA) were placed downstream of the endogenous promoter of rosa26. The Fluc specific shRNA is expressed under the control of the U6- and H1-promoter, respectively, and terminated by five thymidines (shRNA). The loxP-sites flanking the shRNA expression cassettes were used to generate a negative control through cre-mediated recombination. (B) Southern blot analysis of genomic DNA from ES cells containing the targeted insertion of the shRNA (lane 1) or the target configuration (lanes 2 and 3). Homologous recombination at the rosa26 locus is detectable using EcoRV-digested genomic DNA and probe 1, resulting in a 11.7 kb band for the wild type (wt) and a 2.5 kb band for the targeted alleles. E: EcoRV; X: XbaI; neo: FRT-flanked neomycin resistance gene; hyg: FRT-flanked hygromycin resistance gene.

Figure 2

Efficiency of shRNA-mediated Fluc knockdown in mice. (A) Silencing of Fluc under the control of the endogenous rosa26 promoter. Each configuration (control, H1shRNA, U6shRNA) was analyzed using three mice at the age of 6, 12 and 25 weeks, respectively. The efficiency of luciferase knockdown appeared not to be influenced by the age of the animal. Percentages of U6- (white bars) and H1-shRNA-mediated repression of Fluc activity (grey bars) ± standard error of the mean are shown. In the negative controls (black bars), the shRNA expression cassettes were removed through cre-mediated recombination. Relative values of Fluc activity in different organs are given as indicated. All values of Fluc activity were normalized by using the Rluc activity for reference. (B) Comparison of firefly luciferase expression driven by the CAGGS (black bars) and the endogenous rosa26 promoter (white bars), respectively. All values are normalized by measuring the protein content using Bradford assay for reference. (C) Silencing of Fluc under the control of the CAGGS promoter. Percentages of U6-shRNA-mediated repression of Fluc activity (white bars) ± standard error of the mean are shown. In the negative controls (black bars), the shRNA expression cassettes were removed through cre-mediated recombination. Relative values of Fluc activity in different organs are given as indicated. All values of Fluc activity were normalized by using the Rluc activity for reference.

Figure 3

RMCE targeting system for the rosa26 locus. (A) The rosa26 targeting vector comprising zsgreen, PGK-Hyg and CAGGS-FLP was inserted into the rosa26 locus via homologous recombination in ES cells. The F3/FRT sites are oriented in opposite direction to each other. ‘X’ marks the insertion point within the rosa26 locus. The modified locus carrying the RMCE acceptor is called rosa26(RMCE). (B) RMCE by Flp^e-mediated recombination generates the rosa26(RMCE) allele. The exchange vector carries the shRNA expression cassette under the control of the H1 promoter, the F3/FRT pair and a truncated neo^R gene for positive selection. A polyA signal is included to prevent expression of the truncated neo^R gene at random integration sites. (C) Southern blot analysis of genomic DNA from ES cells. The sizes of wt, rosa26(RMCE) and rosa26(RMCE exchanged) are 4.4, 3.9 and 5.8 kb, respectively. In clones 1–3 and 5–9 successful RMCE had occurred. Genomic DNA was digested with HindIII and analyzed using probe 1. X: XbaI, H: HindIII.

Figure 4

Analysis of shRNA-mediated β-galactisidase silencing at the single cell level. Histological sections of organs from wt and CAGGS-lacZ transgenic mice, as well as two CAGGS-lacZ/shRNA transgenic animals are shown as indicated. Cryosections were incubated with X-gal (blue staining) and counterstained with Nuclear Fast Red (red staining). Magnification: 40×.

Figure 5

TaqMan analysis of leptin receptor mRNA in shRNA transgenic versus wild-type control mice. Percentages of leptin receptor mRNA expression in shRNA transgenic mice (grey bars) in comparison with expression levels found in wild-type mice (black bars) ± standard error of the mean are shown. All assays were performed with groups of 4–5 mice at age of 4 months from different organs as indicated.