Mamm Genome (2011) 22:674–684 DOI 10.1007/s00335-011-9356-0 Identification of the translocation breakpoints in the Ts65Dn and Ts1Cje mouse lines: relevance for modeling down syndrome Arnaud Duchon • Matthieu Raveau • Claire Chevalier • Vale´rie Nalesso • Andrew J. Sharp • Yann Herault Received: 11 July 2011 / Accepted: 5 September 2011 / Published online: 28 September 2011 Ó The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Down syndrome (DS) is the most frequent found that the translocations introduce additional aneu- genetic disorder leading to intellectual disabilities and is ploidy in both models, with a monosomy of seven genes in caused by three copies of human chromosome 21. Mouse the most telomeric part of mouse chromosome 12 in the models are widely used to better understand the physio- Ts1Cje and a trisomy of 60 centromeric genes on mouse pathology in DS or to test new therapeutic approaches. The chromosome 17 in the Ts65Dn. Finally, we report here the older and the most widely used mouse models are the tri- overexpression of the newly found aneuploid genes in the somic Ts65Dn and the Ts1Cje mice. They display deficits Ts65Dn heart and we discuss their potential impact on similar to those observed in DS people, such as those in the validity of the DS model. behavior and cognition or in neuronal abnormalities. The Ts65Dn model is currently used for further therapeutic assessment of candidate drugs. In both models, the trisomy Introduction was induced by reciprocal chromosomal translocations that were not further characterized. Using a comparative Down syndrome (DS) remains the most frequent genetic genomic approach, we have been able to locate precisely cause of mental retardation in humans. The genetic basis, the translocation breakpoint in these two models and we trisomy of human chromosome 21 (Hsa21), happens in 1 took advantage of this finding to derive a new and more out of 700–800 births, with a sex ratio of 3 boys for 2 girls efficient Ts65Dn genotyping strategy. Furthermore, we (Huret and Sinet 2000) and it is estimated that there are more than 217,800 cases a year throughout the world (Christianson et al. 2006). This is certainly an underesti- A. Duchon Á M. Raveau Á C. Chevalier Á V. Nalesso Á mation considering that this syndrome accounts for & Y. Herault ( ) approximately 2% of miscarriages (Contestabile et al. Institut de Ge´ne´tique Biologie Mole´culaire et Cellulaire, Translational Medicine and Neuroscience Program, IGBMC, 2010). The clinical picture of this syndrome is extremely CNRS, INSERM, Universite´ de Strasbourg, UMR7104, complex, but the most disabling phenotypes are certainly UMR964, 1 rue Laurent Fries, 67404 Illkirch, France the intellectual disabilities, with delayed learning and e-mail: [email protected] cognition, the appearance of Alzheimer-like disease while A. J. Sharp aging, the morphological anomalies, the locomotor deficit, Department of Genetics and Genomic Sciences, Mount Sinai and cardiac malformations. School of Medicine, 1425 Madison Avenue, Room 14-75B, To improve our knowledge of DS, mouse models have Box 1498, New York, NY 10029, USA been created in recent years. At the genomic level, the long Y. Herault arm of Hsa21 comprises 33.7 Mb of DNA, containing Transgenese et Archivage Animaux Mode`les, TAAM, CNRS, approximately 291 annotated genes in hg19.refGene (Pruitt UPS44, 3B rue de la Fe´rollerie, 45071 Orle´ans, France et al. 2003). Among those genes, 181 have a counterpart in the mouse genome according to the Mammalian Orthology Y. Herault Institut Clinique de la Souris, ICS, 1 rue Laurent Fries, section of the Mouse Genome Informatics (The Jackson 67404 Illkirch, France Laboratory, Bar Harbor, ME). These genes are located in 123 A. Duchon et al.: Identification of the translocation breakpoints 675 three syntenic regions localized on different mouse chro- was recently proposed (Lorenzi et al. 2010). Nevertheless, mosomes (Mmu for Mus musculus). The largest region is classical crossing-over introduces confusion in genotyping found on Mmu16, approximately 37 Mb, including 119 and limits the alternative method to a triage (AD and YH, orthologous genes from Lipi to Zfp295. The next segment, personal communication), not improving the efficiency of including 20 orthologous genes from Umodl1 to Hsf2 bp,is Ts65Dn genotyping. found on Mmu17; and, finally, the most telomeric region, Here we report the characterization of the translocation encompassing 42 orthologous genes between Cstb and breakpoints in both the Ts65Dn and the Ts1Cje line. We Prmt2, is on Mmu10. The order and relative orientation of also propose a new genotyping protocol using classical the genes are well preserved between the two species PCR to facilitate the use of the Ts65Dn model. We further (Dierssen et al. 2009). Several DS murine models were consider the effect of the trisomy of genes located in the developed over the years using controlled chromosomal Ts65Dn Mmu17 region on their expression in one adult engineering techniques (Brault et al. 2006, 2007; Li et al. organ, the heart. Our results will help us better understand 2007; Pereira et al. 2009; Yu et al. 2010a, b). Before that, the translocated chromosomes and open the discussion on the first models selected carried translocations of specific the limits of the use of these DS models compared to the segments homologous to Hsa21. Briefly, the Ts(16C- new engineered mouse lines. tel)1Cje (abbreviated Ts1Cje) was isolated after a targeting experiment aimed at inactivating the Sod1 gene on Mmu16 (Sago et al. 1998). In this line the translocated segment Materials and methods starts upstream of a knockout allele of Sod1 with 67 functional genes and is fused to the telomeric end of Mouse lines Mmu12 (Huang et al. 1997; Pruitt et al. 2003). The Ts(1716)65Dn line (abbreviated Ts65Dn) was isolated after The mice were purchased from the Jackson Laboratory. X-ray irradiation, carrying a segment with 122 genes Both the Ts1Cje line and the Ts65Dn line were kept on a homologous to Hsa21 starting upstream of Mrpl39 and F1 B6C3B genetic background in which the C3B are finishing at the telomeric end of Mmu16, which is trans- sighted C H/HeH, a congenic line for the BALB/c allele at located to a small centromeric part of Mmu17 (Davisson 3 the Pde6b gene (Hoelter et al. 2008). They were bred under et al. 1990). specific pathogen-free (SPF) conditions and were treated in With more than 190 references, Ts65Dn has been compliance with the animal welfare policies of the French extensively studied as a DS mouse model. Notably, Ministry of Agriculture (law 87 848). YH, as the principal Ts65Dn mice show deficits in cognition, learning, and investigator in this study, was granted the accreditation memory, plus additional anomalies related to alterations 67–369 to perform the reported experiments. observed in DS people (Reeves et al. 1995). For example, alterations of Ts65Dn cognitive functions have been cor- related with changes in the number of neurons (Moldrich Genotyping et al. 2007; Roper et al. 2006), decrease in long-term potentiation (LTP), and increase in GABAergic inhibition For both mouse lines, genomic DNA was isolated from tail (Belichenko et al. 2004, 2009; Costa and Grybko 2005; biopsies using a NaCl precipitation technique. Ts1Cje were Kleschevnikov et al. 2004; Popov et al. 2011). Based on genotyped using the protocol described by the Jackson this observation, several treatments were successfully Laboratory (stock No. 004861 JAXÒ Mice Database) by evaluated in Ts65Dn DS models to restore hippocampus- using two pairs of primers. One matches within the neo- dependent learning (Braudeau et al. 2011; Costa et al. mycin gene in Ts1Cje (IMR6916: CTTGGGTGGAG 2008; Fernandez et al. 2007; Moran et al. 2002; Rueda AGGCTATTC and IMR6917: AGGTGAGATGACAGG et al. 2008; Salehi et al. 2009). AGATC) and the other, used as an internal control, mat- However, the exact position of the translocation break- ches in the Tcrd gene (IMR8744: CAAATGTTGCTTG point between Mmu17 and Mmu16 is not precisely defined TCTGGTG and IMR8745: GTCAGTCGAGTGCACAG in the Ts65Dn. Accordingly, the list of genes located on the TTT). centromeric part of Mmu17 in the translocated chromo- Ts65Dn was genotyped using a quantitative PCR pro- some was not precisely known. Thus, the effect of this tocol. The technique is based on the DDCT calculation additional trisomy of Mmu17 genes on DS-related pheno- method between a control gene present in two copies types is difficult to evaluate. In addition, the genotyping is (ApoB) and a target gene present in three copies (Mx1). based mainly on genomic quantification, with modest dis- Two marked probes are used, one for the control gene crimination in the high-throughput process (Liu et al. ApoB: VIC-CCAATGGTCGGGCAC-MGB-NFQÒ, and 2003). An alternative using polymorphic genetic markers another for the target gene Mx1: 6-FAM-CCTGGTCG 123 676 A. Duchon et al.: Identification of the translocation breakpoints CTGTGCA-MGB-NFQÒ (Applied Biosystems, Villebon- (Invitrogen, Cergy Pontoise, France) according to the sur-Yvette, France), coupled with their respective primers manufacturer’s instructions. Sample quality was checked already described (Liu et al. 2003). To avoid sampling using an Agilent 2100 Bioanalyzer (Agilent Technologies, variation, DNA concentration was homogenized and the Massy, France). TaqMan probes were labeled with two different fluorescent reporters (FAM and VIC) so that a multiplexed PCR could Whole-genome expression arrays be used. The qPCR was done in triplicate and we tested a minimum of 15 individuals per experiment. In addition, the Biotinylated cDNAs were prepared from the total RNAs standard deviation of the threshold cycle (CT) triplicates previously mentioned and hybridized onto GeneChip should not exceed the value of 0.3 for each dye in order to Mouse GENE 1.0ST arrays (Affymetrix, Santa Clara, CA, be validated.