A Transient Transgenic RNAi Strategy for Rapid Characterization of Gene Function during Embryonic Development Bryan C. Bjork 1 , Yuko Fujiwara 2. , Shannon W. Davis 3. , Haiyan Qiu 1 , Thomas L. Saunders 3 , Peter Sandy 4¤ , Stuart Orkin 2 , Sally A. Camper 3 , David R. Beier 1 * 1 Genetics Division, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Division of Hematology and Oncology, Children’s Hospital, Harvard Medical School/Howard Hughes Medical Institute, Boston, Massachusetts, United States of America, 3 Departments of Human Genetics and Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America, 4 The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America Abstract RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for ‘‘transient transgenic’’ knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method. Citation: Bjork BC, Fujiwara Y, Davis SW, Qiu H, Saunders TL, et al. (2010) A Transient Transgenic RNAi Strategy for Rapid Characterization of Gene Function during Embryonic Development. PLoS ONE 5(12): e14375. doi:10.1371/journal.pone.0014375 Editor: Ferenc Mueller, University of Birmingham, United Kingdom Received May 14, 2010; Accepted November 24, 2010; Published December 16, 2010 Copyright: ß 2010 Bjork et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Core support was provided by the University of Michigan Cancer Center (National Institutes of Health (NIH) grant CA46592), the University of Michigan Multipurpose Arthritis Center (NIH grant AR20557), the University of Michigan Center for Organogenesis, the University of Michigan Gut Peptide Research Center (NIH grant DK34933), the University of Michigan Nathan Shock Center for the Biology of Aging (NIH grant P30AG013283), and the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000815). SW Davis and SA Camper were supported by NIH grants R01HD34283 and R37HD30428. BC Bjork was supported by F32HD045066 and K12DE014528. BC Bjork, H Qiu, and DR Beier were supported by RO1HD36404 and R01MH081187. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: beier@receptor.med.harvard.edu . These authors contributed equally to this work. ¤ Current address: Constellation Pharmaceuticals, Cambridge, Massachusetts, United States of America Introduction The production of targeted mutations in mice remains the gold standard for the analysis of the loss-of-function studies of specific genes in mammals. However, even with the emergence of large- scale knockout mouse resources, such as those of the International Knockout Mouse Consortium (http://www.knockoutmouse.org/), generation of such mutants using embryonic stem (ES) cells may still require substantial time and resources. In particular, this approach is difficult to pursue for high throughput applications. For instance, linkage and association studies for mutations or strain-specific traits may yield a large number of positional candidate genes, which may require testing individually to assess causality. Similarly, microarray analyses typically result in lists of differentially expressed genes, with little indication regarding which ones may be key regulators. An efficient methodology to rapidly screen genes in vivo would enhance the functional analysis of outputs from high throughput screening. The discovery of RNA interference (RNAi) and its application in mammals has provided a new avenue to study the consequences of reduced gene expression [1,2]. In this process, short 19–25 nt double-stranded RNA (dsRNA) duplexes mediate the degradation of mRNA transcripts that contain an exact match to the dsRNA sequence (reviewed in [3]). This occurs through the recruitment of the RNase III enzyme, Dicer, followed by a multicomponent nuclease complex known as RISC (RNA-induced silencing complex). Alternatively, mismatched dsRNAs can lead to reduced gene activity through the suppression of protein translation [4]. Current methods for the utilization of RNAi as a means to test the effect of loss of gene function involve direct introduction of short interfering RNAs (siRNAs) or expression of precursor short hairpin RNAs (shRNAs) expressed on plasmids and retroviruses [2,5,6]. shRNA-expressing vector systems, including lentivirus and transposable elements vectors, provide highly efficient, stable shRNA expression in cultured cells and transgenic mammals (reviewed in [7,8]). Lentiviral infection of ES cells, morula, or single-cell embryos (via injection into the perivitelline space) has been successfully employed for transgenesis in mice and subsequent RNAi knockdown [9,10]. However, these protocols are not routinely employed in microinjection facilities. In contrast, PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e14375