Simultaneous Cre‐mediated conditional knockdown of two genes in mice

Abstract

One of the most promising techniques to manipulate gene expression in vivo is the use of RNA interference (RNAi). Various approaches were developed to use RNAi in the mouse, including vector-based expression of short hairpin RNAs and the Cre/loxP recombination system. We combined these two approaches to create a vector system that allows the time- and tissue-specific control of two genes at the same time. For this purpose two independent conditional shRNA expression cassettes are combined into a single construct. By the use of different, incompatible pairs of lox sites that flank transcriptional stop cassettes, Cre recombinase can independently activate both short hairpins. Here, we show that Cre simultaneously activates both shRNAs in vitro and in vivo. We applied this technique to silence the widely coexpressed gene pairs Gsk-3alpha/Gsk-3beta and Erk1/Erk2 in murine embryonic stem cells and in the mouse brain.

Phosphorylation and Inactivation of Glycogen Synthase Kinase 3β (GSK3β) by Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A)

“…Simultaneous knockdown of GSK3␣ and GSK3␤ showed a strong decrease in body weight (31), whereas GSK3␤ TG mice that overexpress human GSK3␤ in skeletal muscle showed body weight gain that was due to an increased amount of fat (14). Moreover, a small-molecule GSK3 inhibitor shows anti-obesity effects on HFD-induced obesity as a result of decreased adiposity and improved lipid profiles (25).…”

Citations

“…Combining multiple shRNA cassettes into a single vector is now routine practice, but straightforward systems for using them in conditional knockdown experiments in mice are lacking. Steuber-Buchberger et al address this deficiency in a technology report appearing in genesis , describing coordinated knockdown of two genes in a tissue-specific manner. For each target gene, the authors designed a shRNA sequence for use in the U6 expression cassette; within the hairpin sequence, they introduced an 800-bp Pol III transcription termination signal flanked by lox sites.…”

“…Fortunately, however, mutant loxP sites have been described, and therefore the authors flanked one transcription termination element with wild-type loxP sites and the other with so-called lox2272 sites, restricting possible recombinational events to completely independent excisions. Steuber-Buchberger et al tested two different double knockdown constructs in mice: the first directed against ERK1 and ERK2, and the second against two isoforms of glycogen synthase kinase 3. In each case, mice containing the RNA interference cassettes were bred to a strain that expresses Cre recombinase in neuronal progenitor cells.…”

In Search of the Most Suitable Lentiviral shRNA System

“…Although the idea resulted in a workable system, shRNAs were inefficiently processed [190] and the longer dsRNA molecules are prone to elicit immune responses and off-target effects [195]. Very recently, this approach was used to create a multiplex Cre-LoxP system in which 2 shRNA cassettes are conditionally regulated by an individual fLoxed stuffer fragment [196]. Finally, Coumoul et al [189,197] reported that insertion of the LoxP sites between the DSE and PSE retaining distances did not affect shRNA expression both in vitro and in vivo.…”