Generation of a multipurpose Prdm16 allele by targeted trapping

Gene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays enabling directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1 deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox gene Emx1 demonstrating the utility of the technology for the analysis of tissue-specific gene functions. D is ea se M o de ls & M ec ha ni sm s • D M M • A dv an ce a rt ic le INTRODUCTION To better understand human development and disease, the role of single genes must be examined in the context of the entire organism. Efforts to expand our understanding of molecular mechanisms involved in human development and disease require the systematic analysis of gene function in model organisms such as mice. The availability of the mouse genome sequence showing strong conservation at nucleotide and amino acid levels make mice ideally suited for studying the phenotypic consequences of altered gene expression. A variety of strategies have been employed to introduce specific alterations into the mouse genome. Of these, chemical mutagenesis using N-Ethyl-N-Nitrosourea (ENU) and gene targeting or trapping in mouse embryonic stem (ES) cells have been used extensively in “forward” and “reverse” genetic screens, respectively (Stottmann and Beier, 2010). By exploiting site-specific recombinase systems such as Cre/loxP and Flpe/frt in combination with gene targeting or trapping it became possible to induce spatially and/or temporally controlled mutations in the mouse for functional studies (Branda and Dymecki, 2004; Lao et al., 2012). Gene trapping is an unbiased, high-throughput approach for inducing loss-of-function mutations which is performed with gene trap vectors that simultaneously mutate and report the expression of the trapped gene at the site of insertion (Kitajima and Takeuchi, 1998; Stanford et al., 2001). Classic gene trap vectors comprise a strong 5’ splice acceptor (SA) site, a promoterless reporter and/or selectable marker gene, and a 3’ polyadenylation site which are traditionally introduced into mouse ES cells via electroporation or viral infection to randomly insert throughout the genome. If insertion occurs into an intron of an expressed gene in the correct reading frame, the endogenous transcript is terminated by the gene trap’s polyA site resulting in a fusion transcript from which the reporter is translated along with a truncated and non-functional version of the endogenous protein. Traditional gene traps induce null mutations and require a minimum level of endogenous gene expression for event selection (Friedel and Soriano, 2010; Friedrich and Soriano, 1991). To add conditional features to gene trap vectors, Schnütgen et al. equipped a series of gene trap vectors with flipexcision (FlEx) arrays that enable directional gene trap cassette inversions by Flp and Cre recombinases (Schnütgen et al., 2005). Because these gene traps generate multipurpose alleles enabling a variety of post-insertional modifications, they have been used extensively in combination with gene targeting or trapping by the International Mouse Mutagenesis D is ea se M o de ls & M ec ha ni sm s • D M M • A dv an ce a rt ic le Consortium (IKMC) in an effort to conditionally mutate every protein coding gene of the mouse genome (Bradley et al., 2012). To pursue a gene of interest by taking advantage of the gene trap features to simultaneously inactivate and report gene expression at the insertion site. Friedel et al., used homologous recombination to introduce gene trap cassettes into a prespecified gene, a strategy referred to as “targeted trapping” (Friedel et al., 2005). Here, we describe a mouse transgenic strain carrying a Prdm16 multipurpose allele created by the targeted insertion of the conditional gene trap cassette FlipROSAβgeo* into the second intron of Prdm16. PRDM16 is a transcription factor whose inactivation has severe developmental consequences. It was first identified as an oncogene activated in cases of myelodysplastic syndrome and acute myeloid leukemias (Mochizuki et al., 2000). Loss of function Prdm16 mutations were shown to affect brown adipose cell fate, hematopoietic and neuronal stem cell maintenance and have been linked to the cardiomyopathy developing in patients with 1p36 Deletion syndrome (Aguilo et al., 2011; Arndt et al., 2013; Chuikov et al., 2010; Seale et al., 2008; Seale et al., 2007). Our group and others have demonstrated a role for Prdm16 during mandible and palate development. Its mutational inactivation in mice causes cleft secondary palate (CP) by a similar mechanism as seen in Pierre Robin sequence (PRS)-type clefting (Bjork et al., 2010; Warner et al., 2007). PRS is evident in humans exhibiting micrognathia and glossoptosis which are palate-extrinsic factors that precipitate the development of cleft palate (Tan et al., 2013). We demonstrate that the targeted insertion of the gene trap induces a null mutation that can be rescued and re-induced in pre-specified cells and tissues by consecutive matings to Flpe and Cre deleter mice. By simultaneously mutating and reporting gene expression at the insertion site, the strategy seems ideally suited for the analysis of tissue-specific gene functions at the organismal level. D is ea se M o de ls & M ec ha ni sm s • D M M • A dv an ce a rt ic le