Targeting of Slc25a21 Is Associated with Orofacial Defects and Otitis Media Due to Disrupted Expression of a Neighbouring Gene Simon Maguire 1 , Jeanne Estabel 1 , Neil Ingham 1¤a , Selina Pearson 1 , Edward Ryder 1 , Damian M. Carragher 1¤b , Nicolas Walker 1,2 , Sanger MGP Slc25a21 Project Team 1" , James Bussell 1 , Wai- In Chan 3¤c , Thomas M. Keane 1 , David J. Adams 1 , Cheryl L. Scudamore 4¤d , Christopher J. Lelliott 1 , Ramiro Ramı´rez-Solis 1 , Natasha A. Karp 1 , Karen P. Steel 1¤a , Jacqueline K. White 1 *, Anna-Karin Gerdin 1 1 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom, 2 Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom, 3 Department of Haematology, Cambridge Institute for Medical Research, Cambridge, Cambridgeshire, United Kingdom, 4 Department of Pathology and Infectious Diseases, Royal Veterinary College, Hatfield, Hertfordshire, United Kingdom Abstract Homozygosity for Slc25a21 tm1a(KOMP)Wtsi results in mice exhibiting orofacial abnormalities, alterations in carpal and rugae structures, hearing impairment and inflammation in the middle ear. In humans it has been hypothesised that the 2- oxoadipate mitochondrial carrier coded by SLC25A21 may be involved in the disease 2-oxoadipate acidaemia. Unexpectedly, no 2-oxoadipate acidaemia-like symptoms were observed in animals homozygous for Slc25a21 tm1a(KOMP)Wtsi despite confirmation that this allele reduces Slc25a21 expression by 71.3%. To study the complete knockout, an allelic series was generated using the loxP and FRT sites typical of a Knockout Mouse Project allele. After removal of the critical exon and neomycin selection cassette, Slc25a21 knockout mice homozygous for the Slc25a21 tm1b(KOMP)Wtsi and Slc25a21 tm1d(KOMP)Wtsi alleles were phenotypically indistinguishable from wild-type. This led us to explore the genomic environment of Slc25a21 and to discover that expression of Pax9, located 39 of the target gene, was reduced in homozygous Slc25a21 tm1a(KOMP)Wtsi mice. We hypothesize that the presence of the selection cassette is the cause of the down regulation of Pax9 observed. The phenotypes we observed in homozygous Slc25a21 tm1a(KOMP)Wtsi mice were broadly consistent with a hypomorphic Pax9 allele with the exception of otitis media and hearing impairment which may be a novel consequence of Pax9 down regulation. We explore the ramifications associated with this particular targeted mutation and emphasise the need to interpret phenotypes taking into consideration all potential underlying genetic mechanisms. Citation: Maguire S, Estabel J, Ingham N, Pearson S, Ryder E, et al. (2014) Targeting of Slc25a21 Is Associated with Orofacial Defects and Otitis Media Due to Disrupted Expression of a Neighbouring Gene. PLoS ONE 9(3): e91807. doi:10.1371/journal.pone.0091807 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received November 28, 2013; Accepted February 13, 2014; Published March 18, 2014 Copyright: ß 2014 Maguire 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: This work was supported by the Wellcome Trust (grant no 098051 to WTSI) (http://www.wellcome.ac.uk/) and the European Union’s Seventh Framework program under grant agreement number 282510 - BLUEPRINT (NW) (www.blueprint-epigenome.eu/). 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: jkw@sanger.ac.uk ¤a Current address: Wolfson Centre for Age-Related Diseases, King’s College London, Guy’s Campus, London, United Kingdom ¤b Current address: Medical Research Council National Institute for Medical Research, Mill Hill, London, United Kingdom ¤c Current address: State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau ¤d Current address: Medical Research Council Harwell, Harwell Science and Innovation Campus, Oxford, Oxfordshire, United Kingdom " Membership of the Sanger MGP Slc25a21 Project Team is provided in the Acknowledgments. Introduction Knockout mice are invaluable tools for studying the function of genes both during embryonic development and in the adult. Classically, gene disruption in mice is achieved by replacing a part of the target gene with a selectable marker, e.g. a neo cassette, leading to constitutive ablation of the targeted gene. More contemporary approaches include the creation of conditional alleles and targeted gene traps, as well as small hairpin RNA (shRNA) and the use of lentiviral transgenesis, reviewed in [1]. The Sanger Institute Mouse Genetics Project [2] is contributing to an international effort to conduct systematic, large-scale gene function analysis in a mammalian system, through the generation of mice using targeted ES cells available from the EUCOMM and KOMP resources [3]. The typical EUCOMM and KOMP knockout first conditional ready targeted trap [tm1a(EUCOMM) and tm1a(KOMP)] is a powerful allele configured with the potential to convert to a conditional allele or a lacZ tagged null allele [3]. This tm1a allele was selected for pragmatic reasons, including there being no requirement for additional breeding to modify the allele, as targeted traps typically disrupt expression of the targeted gene [3,4], and the major advantage that the allele, with full potential for conversion, would be cryopreserved for subsequent community use. However, it has previously been reported that the presence of selectable markers used for gene targeting, such as the neo cassette in the EUCOMM and KOMP PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e91807