Transgenic RNAi offers a flexible and systematic alternative to homologous recombination for studying gene function in vivo.
While RNAi cannot replace gene targeting in creating precise genetic lesions, it enables fast, reproducible, and cost-effective generation of transgenic mouse models with unique capabilities for spatial, temporal, and reversible suppression of endogenous genes. Here we set out to optimize every step in the production and analysis of shRNA-transgenic mice by developing: 1) a system that enables efficient ES cell targeting, enhanced gene silencing, and fluorescence-coupled tracking of shRNA expression in vivo
and 2) an approach to rapidly combine shRNAs with existing disease alleles. Importantly, the system is modular such that it can be modified to incorporate alternate reporters (eg. turboRFP, mCherry), tandem shRNA cassettes, or be adapted for controlled expression of additional non-coding RNA species (unpublished data). Additionally, we have recently developed a systematic and high-throughput screening methodology to identify the most efficient shRNAs targeting any given transcript (Fellmann et al., in press
); these shRNAs can then be seamlessly incorporated into the production of shRNA transgenic mice. Together with the availability of optimized RNAi triggers, the system provides a high throughput platform that streamlines production of ES cells and transgenic mice harboring conditional shRNAs, enabling exquisite control of endogenous gene expression in vivo
RNAi-mediated gene silencing does not produce a complete null situation and hence shRNA transgenics will not replace the need for conditional knockout mice, at least in some settings. Rather, we envision that this technology will provide a complimentary approach when speed and scale are desirable – a situation that may be increasing as biologists try to understand the avalanche of information emerging from various genome projects. We also envision that our system will enable the evaluation of hypomorphic phenotypes reflective of many human conditions and disease states and may be applicable to the examination of potential drug targets and in this context, the level and duration of gene suppression that can be tolerated in vivo. Here we demonstrate the strength of our system for investigating gene function in vitro and in vivo during multiple stages of embryonic and postnatal development and disease progression. Importantly, the ability to reversibly regulate gene expression allowed us to explore the consequences of transient APC loss during development and define a small window of development where APC is essential for regulating skeletal morphogenesis.
Using single and multi-allelic approaches, we also showed how shRNA transgenic mice can be used to accurately model human disease and, through gene reactivation, assess the potential of therapeutic targeting strategies. Indeed, the system provides the unique capability of genetically mimicking the action of potential drugs whose action would likely be transient and incomplete, enabling validation of drug targets and characterization of drug toxicities in a manner that has not previously been possible. Finally, further application of the multi-allelic ‘speedy’ approach should provide a relatively high throughput platform for in vivo characterization of candidate genes identified using shRNA-based screens or genome-wide association studies and will facilitate triaging and prioritizing drug targets in expensive drug development programs.
Despite the advances shown here, the limited scope and effectiveness of available tet-transactivator strains and the paucity of shRNAs that act efficiently at single copy represent two remaining barriers to achieving an ideal system. As a first step towards addressing the former issue, we developed a tet-transactivator strain (CAG-rtTA3) that provides stronger and more widespread expression of TRE-regulated shRNAs than any other strain we have tested, including R26-rtTA, Actin-rtTA and CMV-rtTA (data not shown). Although some organs, such as the brain, spleen and lung showed lower GFP expression and reduced luciferase knockdown (see Figure S4
), we believe this is likely due to limited delivery of DOX to these cells (in the case of brain) or sub-optimal rtTA expression rather than inaccessibility of the targeted ColA1
locus. In fact, tissue-specific expression of tet-transactivators in CCSP-rtTA and Vav-tTA strains can promote effective ColA1
-targeted shRNA expression in lung epithelium () and B-cells (data not shown), respectively. It will be important to determine which of the >100 existing tet-transactivator strains (http://www.zmg.uni-mainz.de
) will provide sufficient tTA/rtTA expression for effective knockdown and to develop additional robust and versatile strains.
We believe that transgenic systems such as the one described here have the potential to transform mammalian genetics. Indeed, in lower organisms such as D. melanogaster and C. elegans, the application of in vivo RNAi on a broad scale has had an enormous impact on the ability of these models to provide insights into the physiology of whole organisms in isolation or in the context of other disease associated alleles. With similar capabilities now achievable in mice, it should be possible to rapidly explore all aspects of mammalian physiology and provide clues to the molecular control of normal development, aging, and disease. Accordingly, we have now implemented a 96-well targeting format to enable paralleled production of hundreds of ES cells and transgenic mice with inducible and reversible, RNAi-mediated gene silencing. With this pipeline it is now possible and indeed feasible, to develop single and multi-allelic shRNA transgenic animals to evaluate in parallel the function of many mammalian genes.