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Human Molecular Genetics Advance Access originally published online on April 5, 2007
Human Molecular Genetics 2007 16(11):1359-1366; doi:10.1093/hmg/ddm086
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Duplication of the entire 22.9 Mb human chromosome 21 syntenic region on mouse chromosome 16 causes cardiovascular and gastrointestinal abnormalities

Zhongyou Li1,{dagger}, Tao Yu1,{dagger}, Masae Morishima3,{ddagger}, Annie Pao1, Jeffrey LaDuca1, Jeffrey Conroy1, Norma Nowak1,2, Sei-Ichi Matsui1, Isao Shiraishi3 and Y. Eugene Yu1,2,*

1 Department of Cancer Genetics and Center for Genetics and Pharmacology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA, 2 New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14263, USA and 3 Department of Pediatric Cardiology and Nephrology, Kyoto Prefectural University of Medicine, Kamigyo, Kyoto 602-8566, Japan

* To whom correspondence should be addressed at: Department of Cancer Genetics and Center for Genetics and Pharmacology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. Tel: +1 7168451099; Fax: +1 7168451698; Email: yuejin.yu{at}roswellpark.org

Received December 29, 2006; Accepted March 31, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Down syndrome is caused by a genomic imbalance of human chromosome 21 which is mainly observed as trisomy 21. The regions on human chromosome 21 are syntenically conserved in three regions on mouse chromosomes 10, 16 and 17. Ts65Dn mice, the most widely used model for Down syndrome, are trisomic for ~56.5% of the human chromosome 21 syntenic region on mouse chromosome 16. To generate a more complete trisomic mouse model of Down syndrome, we have established a 22.9 Mb duplication spanning the entire human chromosome 21 syntenic region on mouse chromosome 16 in mice using Cre/loxP-mediated long-range chromosome engineering. The presence of the intact duplication in mice was confirmed by fluorescent in situ hybridization and BAC-based array comparative genomic hybridization. The expression levels of the genes within the duplication interval reflect gene-dosage effects in the mutant mice. The cardiovascular and gastrointestinal phenotypes of the mouse model were similar to those of patients with Down syndrome. This new mouse model represents a powerful tool to further understand the molecular and cellular mechanisms of Down syndrome.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Down syndrome is a major human chromosome disorder reflected as a duplication imbalance of chromosome 21 and is most frequently caused by the presence of an extra copy of the entire human chromosome 21 in patients (1). Down syndrome is the most frequent live-born human aneuploidy, occurring in approximately one out of 800–1000 live births (2,3). The phenotypes of Down syndrome include heart defects, craniofacial abnormalities, gastrointestinal anomalies, mental retardation, leukemia and Alzheimer's disease with variable penetrance and onsets (4,5). The mouse has been used extensively for modeling Down syndrome because the genomic segments on three mouse chromosomes, i.e. chromosomes 10 (~39 orthologous genes), 16 (~113 orthologous genes) and 17 (~19 orthologous genes), are syntenic to the regions on human chromosome 21 (Fig. 1) (6). As currently the most popular mouse model of Down syndrome, the Ts65Dn strain was developed by analyzing the chromosomal rearrangements that were randomly induced by irradiation and is trisomic for ~13 Mb in the human chromosome 21 syntenic region on mouse chromosome 16. But it is also trisomic for a subcentromeric region of mouse chromosome 17, which is not syntenic to any region on human chromosome 21 (7). The Tc1 trans-species mouse strain, carrying an almost entire human chromosome 21, has recently been developed using irradiation microcell-mediated chromosome transfer (8), which exhibits several key phenotypes of Down syndrome. However, the random loss of this human chromosomal fragment during mouse development resulted in variable levels of mosaicism of the extra chromosome in different tissues, confounding the analysis of phenotypic consequences.


Figure 1
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  		Figure 1. Schematic of the syntenic segments in the human 21q11–q22.3 region and in the distal region of mouse chromosome 16. Three genes are shown, including those at the ends of the syntenic segments.

 
Development of the Cre/loxP-mediated mouse chromosome engineering methodology has resulted in mouse models that are more representative and genuine with regard to the genotypes and phenotypes observed in human chromosomal disorders. This technology enables us to introduce defined chromosomal rearrangements, such as deletions, duplications, inversions and translocations, into the mouse genome by engineering them in embryonic stem (ES) cells. Mouse strains have been developed using chromosome engineering to model the human chromosomal rearrangements that are responsible for DiGeorge syndrome (9–11), Prader–Willi syndrome (12), Smith–Magenis syndrome (13), as well as Down syndrome (14). Chromosomal disorders are very difficult to analyze in humans because one must rely on rare rearrangements to subclassify the phenotype. In contrast, specific sub-deletions or sub-duplications can be generated in mice, enabling specific associations to be drawn between aspects of the phenotype and genes in the rearranged region. Indeed, this approach was instrumental in the identification of the causative gene for the principal cardiovascular defect in DiGeorge syndrome (10,11,15).

To establish a more genuine mouse model reflective of human Down syndrome, we generated a 22.9 Mb duplication in mice, which spans the entire human chromosome 21 syntenic region on mouse chromosome 16. Phenotypic analysis showed that the duplication was responsible for heart defects and gastrointestinal anomalies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Characterization of translocation breakpoint in Ts65Dn mice
Ts65Dn strain is currently the most popular mouse model of Down syndrome and is segmentally trisomic from Mrpl39 to the telomere on mouse chromosome 16 (Fig. 1) (7,16). However, Ts65Dn mice are also trisomic for a >4.8 Mb subcentromeric region of mouse chromosome 17, which is not syntenic to any region on human chromosome 21 (7). We further defined the translocation breakpoint in Ts65Dn mice using fluorescent in situ hybridization (FISH) analysis and found that this mouse chromosome 17 trisomic region is actually >5.8 Mb and contains as many as 19 genes, including Synj2 (Fig. 2) (www.ensembl.org). Therefore, the possibility cannot be excluded that this trisomic region may contribute to the mutant phenotypes of Ts65Dn mice.


Figure 2
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  		Figure 2. The FISH analysis of metaphase chromosomes of splenocytes isolated from a Ts65Dn mouse. (A) Arrowhead: the hybridization signal of BAC RP23-147G23, indicating the Ts65Dn translocation chromosome contains the genomic region surrounding the Synj2 gene. (B) Higher magnification of area boxed in A. (C) Schematic of the genomic locations of BAC probes and Synj2.

 
Generation of Dp (16)1Yu in mice
The D930038D03Rik and Zfp295 genes are located at the proximal and distal ends of a 22.9 Mb region on mouse chromosome 16, respectively, that is syntenic to human 21q11q22.3 (Fig. 1) (17). This mouse syntenic region contains approximately 113 orthologous genes (Supplementary Material, Table S1) (6). A chromosomal duplication in this region was generated using long-range Cre/loxP-mediated recombination, as described in Materials and Methods (Figs 1 and 3).


Figure 3
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  		Figure 3. Generation of the chromosomal rearrangements. (A) Strategy to generate the chromosomal rearrangements based on Cre/loxP-mediated recombination in the G1 or G2 phase of the cell cycle (19). A, AflII; N, NheI; 3', 3'-Hprt; 5', 5'-Hprt; Ag, K14-Aguoti gene; Ty, tyrosinase minigene; Puro, puromycin-resistance gene; Neo, neomycin-resistance gene; arrowhead, loxP site. (B) Southern blot analysis of the samples of ES cell DNA that were digested with AflII. ES cell DNA that contained the duplication and the reciprocal deletion (lane 1) and ES cell DNA containing the duplication and the targeted chromosome (lane 2) were hybridized to probe C. (C) FISH analysis of metaphase chromosomes prepared from the embryonic fibroblasts carrying Dp(16)1Yu/+. (a and b) The embryonic fibroblasts isolated from the embryos generated from a chimeric male; (c and d) the embryonic fibroblasts isolated from F3 Dp(16)1Yu/+ embryos in the 129Sv background. (a) The RP23-81D13 BAC probe (green) hybridized to the EphB3 marker, which is located outside the region, whereas the RP23-264F8 BAC probe (red) hybridized to the Zfp294 marker located between D930038D03Rik and Zfp295. (b) Higher magnification of the area boxed in (a). (c) The RP23-65F24 BAC probe (red) hybridized to the region between Zfp295 and Zfp294, whereas RP23-185P17 probe (green) hybridized to the region between Zfp294 and D930038D03Rik. (d) Higher magnification of the area boxed in (c). (D) Array CGH analysis of DNA isolated from a Dp(16)1Yu/+ mouse. (a) Whole-genome array CGH profile and (b) CGH profile of the mouse chromosome 16. Plotted are log 2-transformed hybridization ratios of Dp(16)1Yu/+ mouse DNA versus control DNA. The BAC clones spanning the duplication region (green dots) are denoted.

 
AB2.2 ES cells were sequentially targeted with pTVD930038D03Rik and pTVZfp295 (Figs 1 and 3A). Two double-targeted clones were isolated which were transiently transfected with a Cre-expression vector, and ES cell clones that had undergone loxP recombination and activated the Hprt selection cassette were selected in hypoxanthine, aminopterin and thymidine (HAT) medium. Sib selection of the two HAT-resistant clones indicated that clone 1D1 was G418-sensitive and puromycin-resistant, whereas clone 1D2 was resistant to both G418 and puromycin. These results suggested that 1D1 carried a duplication, whereas clone 1D2 carried both the deletion and the duplication (18,19), which were confirmed by Southern blot analysis (Fig. 3B). We used these ES cell clones to generate chimeras. The germline transmission of the duplication after crossing 129Sv females with chimeric males was confirmed by Southern blot analysis as well as FISH analysis (Fig. 3Ca and b). To further confirm the presence of the duplication spanning the entire syntenic region, we performed BAC array-based comparative genomic hybridization (aCGH) with genomic DNA isolated from a Dp(16)1Yu/+ mouse. As shown in Figure 3D, mouse array CGH precisely detected a single copy duplication located between 75.2 and 98.1 Mb on mouse chromosome 16, by 39 BAC clones (green dots). The duplication was designated as Dup (16)(D930038D03Rik-Zfp295)1Yu, abbreviated as Dp(16)1Yu. The viable Dp(16)1Yu/+ embryos at E18.5 were present at normal Mendelian ratios, but only ~38% of Dp(16)1Yu/+ mice survived at weaning. The surviving mutant mice are overtly normal and have reached ~10 months of age. These Dp(16)1Yu/+ mice were crossed to wild-type 129Sv mice for two generations, and the transmission of the intact Dp(16)1Yu was confirmed by FISH analysis of the F3 embryonic fibroblasts (Fig. 3Cc and d). Since BAC probes RP23-185P17 and RP23-65F24 used in the FISH analysis are mapped near D930038D03Rik and Zfp295, respectively, but within the duplication interval, the duplications of the signals hybridized to both probes in Figure 3Cc and d provided the evidence that the entire duplicated region of Dp(16)1Yu is retained in the mutant F3 embryos.

Elevated expression levels of the duplicated genes
We used Taqman real-time quantitative PCR to compare the mRNA levels for the genes located within the duplicated interval in mice carrying different genotypes. Gapdh is located on mouse chromosome 6 and served as a reference gene of the disomic state in the Dp(16)1Yu/+ and +/+ mice. The analysis of several genes located within Dp(16)1Yu in the brain and heart tissues showed that the segmental trisomy altered the transcript levels of the genes in the brain and heart of the Dp(16)1Yu/+ model (Table 1), reflecting the dosage imbalance for the duplicated region. This result supports the conclusion that the duplicated genes are expressed with the exception for transcriptionally inactive genes. Dscam did not express in the heart in Dp(16)1Yu/+ mice, which is consistent with the result obtained from Ts65Dn mice (16).


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  		Table 1. Normalized relative values (RQ) of expressiona

 
Heart defects and gastrointestinal anomalies caused by Dp(16)1Yu
Cardiovascular and gastrointestinal abnormalities are the leading neonatal problems that require surgical interventions to prevent early death among patients with Down syndrome (4,20). We therefore examined the cardiovascular and gastrointestinal systems of the Dp(16)1Yu/+ embryos at E18.5, since heart defects and gastrointestinal anomalies may contribute to the poor survival of Df(16)1Yu/+ mice after birth. The morphological analysis revealed that ~37% of Dp(16)1Yu/+ embryos (n = 30) exhibited structural heart defects (Table 2). Two of them exhibited two or more abnormalities that are characteristics of the tetralogy of Fallot (TOF) complex, including perimembranous ventricular septal defects (Fig. 4I and J), overriding of the aorta (Fig. 4I and J) and narrowed outflow track of the right ventricle (Fig. 4I and J). We also observed ventricular septal defects, atrial septal defects, cleft mitral valves, severe coarctation of the aorta and double outlet right ventricle (Fig. 4, Table 2). These malformations of the cardiovascular system are similar to those observed in patients with Down syndrome (20). The morphological analysis also revealed that ~26 and 22% of Dp(16)1Yu/+ embryos exhibited annular pancreas and malrotation of the intestine, respectively (Fig. 5). Heart defects or gastrointestinal anomalies were not detectable in 30 wild-type embryos at the same embryonic stage from control matings.


Figure 4
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  		Figure 4. Cardiovascular anomalies in Dp (16)1Yu/+ embryos at E18.5. (B, D and F) show a Dp(16)1Yu/+ embryo with multiple anomalies in the cardiovascular system. (A, C and E) show a wild-type control. Intracardiac views of atria and great arteries after removal of the ventricles (A and B) reveal an ASD (blue arrow head) and coarctation of the aorta (blue arrow) (B). Superior views of atrioventricular valves after removal of the free walls (C and D) shows the cleft of the mitral valve (blue arrow head), indicating developmental defect of atrioventricular valve (D). Intracardiac views of the right ventricles from the ventral side after removal of the free wall (E and F) shows double outlet right ventricle with perimembranous VSD (F). (G) shows the normal ventricle of a wild-type embryo with intact ventricular septum and wide outflow tract (blue arrows). (H–J) shows the intracardiac views of the right ventricle of a Dp(16)1Yu/+ embryo from the right side after removal of the free wall. (H) shows a conal VSD with the aortic orifice located behind a ridge of the VSD foramen. (I) shows a TOF-like group of defects. An abnormal deviation of the parietal band (asterisk) is associated with VSD (blue arrow heads), causing the overriding of the aorta. Note that because of the overriding of the aorta, the aortic (Ao) valve is visible through the VSD. (J) shows the same ventricle in (I) from a different angle, revealing a narrowed outflow tract of the right ventricle [blue arrows, compared with the outflow track in (G)] due to the obstruction caused by the deviation of the parietal band. Ao, aorta; At Sept, atrial septum; D, ductus arteriosus; LAA, left atrial appendage; LV, left ventricle; MPA, main pulmonary artery; MV, mitral valve; PA, pulmonary artery; PV, pulmonary vein; RAA, right atrial appendage; RV, right ventricle; TV, tricuspid valve. ASD, atrial septal defect. VSD; ventricular septal defect. Scale bar, 1 mm.

 


Figure 5
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  		Figure 5. Gastrointestinal anomalies in Dp(16)1Yu/+ embryos at E18.5. The ventral (B) and superior (D) views show annular pancreas (blue arrows) and malrotation of the intestine in a Dp (16)1Yu/+ embryo. (A and C) Ventral and superior views of a wild-type control, respectively. P, pancreas; D, duodenum; E, esophagus; St, stomach; Sp, spleen. Scale bar, 2 mm.

 


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  		Table 2. Cardiovascular anomalies in Dp(16)1Yu/+ embryos at E18.5

 
Since the targeted regions by the targeting vectors pTVD930038D03Rik and pTVZfp295 are located ~174 kb proximal and 24 kb distal from the structural genes of D930038D03Rik and Zfp295, respectively, the targeting events did not generate mutated forms of the proteins by disrupting the structures of the genes. Although it is formally possible that the targeted alleles may alter the expression levels of the genes by affecting regulatory elements in the regions, heterozygous and homozygous mutant mice carrying either targeted allele did not exhibit any mutant phenotype, suggesting the mutant phenotypes observed in the Dp(16)1Yu/+ model are not caused by the targeting events at the two endpoints but by the presence of the duplication.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We have generated a new mouse model for Down syndrome carrying a 22.9 Mb duplication, which, to our knowledge, is the largest duplication established in mice. The duplicated mouse genomic region is syntenic to a segment on human 21q11.2–q22.3, which spans the entire human chromosome 21 syntenic region on mouse chromosome 16 (Fig. 1).

Down syndrome is the most common chromosomal disorder that causes congenital heart disease. Heart defects are detected in 40–60% of individuals with Down syndrome (4,21–23). The frequent cardiac anomalies associated with Down syndrome are atrioventricular septal defect (23–45% of newborns with Down syndrome), ventricular septal defect (33–35%) and atrial septal defect (8–21%). Other lesions occur at relatively lower frequency: TOF, cleft of the mitral valve, coarctation of the aorta and double outlet right ventricle (4,21,23). The trisomy 16 mice carrying a complete extra copy of mouse chromosome 16 were the first trisomic model for heart defects in Down syndrome. Although various aspects of heart defects were observed in the trisomy 16 mouse embryos, it was difficult to establish the causative role for the human chromosome 21 syntenic region, since only ~23.3% of the mouse chromosome 16 genomic region is syntenic to a region on human 21q11.2–q22.3 and the remaining regions of mouse chromosome 16 are syntenic to the regions on human 22q11, 16p and 3q (24–26). Atrioventricular septal defects and ventricular septal defects have been identified in the Tc1 trans-species mice (8). Ventricular septal defects have also been identified recently in ~8% of Ts65Dn mice (27). However, the presence of the non-syntenic trisomic segment of mouse chromosome 17 in Ts65Dn mice and the mosaicism of Tc1 mice complicated the interpretation of the genotype-phenotype correlation. As a new model, the Dp(16)1Yu/+ mutants exhibit a wide spectrum of cardiovascular anomalies, mimicking the phenotypes of Down syndrome. When compared with Ts65Dn and Tc1 mice, the Dp(16)1Yu/+ model offers a unique advantage for being a constitutional genomic alteration and trisomic solely for the human 21q11.2–q22.3 syntenic region.

Down syndrome is a leading cause of gastrointestinal anomalies and results in ~430- and 44-fold increased risks in annular pancreas and intestinal malrotation, respectively (20,28). Annular pancreas is the most common developmental abnormality of pancreas, which may lead to duodenal stenosis. The molecular mechanisms underlying these structural anomalies are unknown at present. These gastrointestinal phenotypes have been consistently observed in Dp(16)1Yu/+ embryos, but have not been detected in any other mouse models of Down syndrome. Therefore, Dp(16)1Yu/+ mice represent a unique model for gastrointestinal anomalies in Down syndrome.

Many causative genes have been identified for heart defects (29,30), whereas only two genes are known to be associated with annular pancreas and malrotation of the intestine (31,32). However, none of these known genes are located on human chromosome 21. Close similarities in the phenotypes of patients with Down syndrome and Dp(16)1Yu/+ embryos suggest that duplication of one or more genes in the syntenic region may be responsible for the abnormalities in cardiovascular and gastrointestinal development for both species. The identification of a trisomic region of the mouse genome that is responsible for heart defects and gastrointestinal anomalies will greatly facilitate the effort to identify minimal critical regions and eventually specific genes that cause these diseases. The minimal critical regions could be identified by generating and analyzing new mouse strains carrying sub-duplications within the syntenic region on muse chromosome 16. The generation of sub-duplications has been made simpler by the newly established Mouse Insertional and Chromosome Engineering Resource, which contains ~100,000 chromosome-engineering vectors distributed throughout the mouse genome (33). If a causative gene that is responsible for heart defects and/or gastrointestinal anomalies can be located in a sub-duplication of ~1 Mb, a complementary strategy of BAC transgenics could be used to map the gene to a single BAC (34,35). Finally, compound mutant mice may be generated carrying duplication or a BAC and a knockout of a candidate gene identified by BAC transgenics. The causative role for a candidate gene could be established based on the elimination of a duplication- or BAC-associated phenotype by the knockout allele in the compound mutant. The knockout alleles generated from on-going public knockout mouse projects will greatly facilitate such a gene-hunting strategy (36,37).

Modeling human genomic abnormalities in mice has become an essential approach for the study of chromosomal disorders of humans. This approach has provided an important strategy for isolating the genes that are responsible for the clinical features associated with the chromosomal disorders of humans. The Dp(16)1Yu/+ mice are a model for human cardiovascular defects and gastrointestinal anomalies associated with Down syndrome. This model should serve as a powerful tool for the eventual identification of the gene or genes that are responsible for these Down syndrome-associated phenotypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Gene targeting in ES cells
The targeting vectors pTVD930038D03Rik and pTVZfp295 were from the 3'-Hprt and the 5'-Hprt libraries, respectively (38). The vectors from both libraries contain inserts of DNA that were generated by partial Sau3AI digestion of genomic DNA from the 129S5 mouse strain. The mouse genomic inserts of pTVD930038D03Rik and pTVZfp295 are mapped to the regions immediately proximal to D930038D03Rik and distal to Zfp295, respectively.

We carried out culturing of the AB2.2 line of ES cells, electroporation and Southern blot analysis as described previously (39). Prior to electroporation, pTVD930038D03Rik and pTVZfp295 were linearized in the homology regions. Targeted ES cell clones were identified by Southern blot with PCR products as probe A or B, which map to the regions external to the homology regions of the targeting vectors.

Generation of chromosomal rearrangements in ES cells and mice
The pOG231 Cre-expression vector (40) was electroporated into double-targeted clones, and ES cell clones with recombined products were selected in HAT medium as described previously (41,42). Germline-transmitting chimeras were generated from ES cell lines carrying the engineered chromosomes by microinjection of blastocysts isolated from albino C57B6/J-Tyrc-Brd females as described previously (43).

Fluorescent in situ hybridization
Chromosome spreads of embryonic fibroblasts and splenocytes were performed as described previously (44). BAC clones were used as probes for FISH (45). To detect the duplication between D930038D03Rik and Zfp295, BAC clones RP23-264F8 and RP23-65F24 were labeled with biotin and detected with rhodamine–avidin. BAC clones RP23-81D13 and RP23-185P17 were labeled with digoxigenin and detected with fluorescin isothiocyanate-antidigoxigenin antibody. RP23-264F8 contains Zfp294, which was confirmed by sequencing the PCR product amplified with gene specific primers 5'-GAGCGAGACCCTGGAGCTGTTT-3' (forward) and 5'-AGACTGAGCCCCTCCGTCAGAG-3' (reverse). Zfp294 is located 11.1 Mb proximal to Zfp295 within the rearranged interval. RP23-81D13 contains EphB3 gene, which was confirmed by sequencing the PCR product amplified with gene-specific primers 5'-CACCCCCGGGGTGTGATCTCCA-3' (forward) and 5'-TCAGGCCCAGCTGCCGAGGTA-3' (reverse). EphB3 is located 54 Mb proximal to D930038D03Rik. RP23- 185P17 is mapped 1.1 Mb distal to D930038D03Rik, whereas RP23-65F24 is mapped 0.5 Mb proximal to Zfp295. To further define the translocation breakpoint in Ts65Dn mice, BAC clone RP23-3G4 was labeled with biotin and detected with rhodamine–avidin. BAC clone RP23-147G23 was labeled with digoxigenin and detected with fluorescin isothiocyanate-antidigoxigenin antibody. RP23-147G23 which contains Synj2 and RP23-3G4 are mapped to 5.8 and 11.3 Mb from the centromere of mouse chromosome 17, respectively. Chromosomes were counterstained with 4',6'-diamidino-2-phenylindole.

Array-based comparative genomic hybridization
Genomic DNA was prepared from the tail tissue of a male Dp(16)1Yu/+ mouse using DNeasy Tissue Kit (Qiagen). Genomic DNA isolated from a wild-type 129Sv female mouse was used as the reference control for BAC aCGH analysis. Control and Dp(16)1Yu/+ genomic DNA (1 µg each) was fluorescently labeled using the BioArray CGH Labeling System (Enzo Life Sciences) for 4 h as per manufacturer's instructions. The test and reference probes were purified, combined with 100 µg mouse Cot-1 (Invitrogen) and ethanol precipitated. Following centrifugation, the probes were resuspended in 110 µl SlideHyb Buffer #3 (Ambion) containing 5 µl of 100 µg/µl Yeast tRNA (Invitrogen), heated to 95°C for 5 min and incubated for 30 min at 37°C to block repetitive elements on the probe (46). A genome-wide sequence-anchored mouse BAC array was generated by LM-PCR essentially as described (47–50). Each array contains ~6500 RPCI-23 and -24 BAC clones spotted in triplicate, representing ~0.5 Mb genomic resolution. A complete list of the BAC clones spotted on the 6K mouse array can be found at http://genomics.roswellpark.org. Hybridization to the BAC array was performed for 16 h at 55°C using a GeneMachine hybridization station (Genomic Solutions, Inc.). The hybridized aCGH slides were scanned for both Cy3 (test) and Cy5 (control) channels. Image analysis was performed using ImaGene (version 7.0.1) software from BioDiscovery, Inc. The log2 ratios of the test/control were normalized using a subgrid loess with the clones on the sex chromosome given a weight of 0. Mapping information was added to the resulting normalized log2 test/control values. The mm8 mapping data for each BAC was found by querying the mouse genome sequence at http://genome.ucsc.edu.

Real-time quantitative PCR
Taqman real-time quantitative PCR was used to compare the RNA levels of the genes located within the rearranged interval in mice carrying different genotypes. Gapdh, located on mouse chromosome 6, was used as the internal disomic control for all of the mice examined. Total RNAs were isolated from mouse brains and hearts using TRIzol Reagent (Invitrogen). One microgram of the pooled RNA from three mice with the same genotype was used to generate cDNA by using Superscript version III reverse transcriptase (Invitrogen). The specific primers and probes of the genes were obtained from the TagMan® Gene Expression Assays System of Applied Biosystems, Inc. A 0.5 µg of cDNA from each genotype was analyzed by ABI 7900HT Real-Time Thermocycler (Applied Biosystems) with the following amplification conditions: an initial activation and denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, primer annealing and extension at 60°C for 1 min.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The authors thank P. Szurek and D. McQuaid for their assistance. This work was supported by grants from Roswell Park Alliance Foundation, Louis Sklarow Memorial Fund, Association for Research of Childhood Cancer, American Cancer Society and Jerome Lejeune Foundation (Y.E.Y.), and in part by the Roswell Park Cancer Institute Cancer Center Support Grant CA 16056.

Conflict of Interest statement. None of the authors has any conflict of interest.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back

{ddagger} Present address: International Research and Educational Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Tokyo 162-8666, Japan. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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F. K. Wiseman, K. A. Alford, V. L.J. Tybulewicz, and E. M.C. Fisher
Down syndrome--recent progress and future prospects
Hum. Mol. Genet., April 15, 2009; 18(R1): R75 - R83.
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