Human Molecular Genetics, 2001, Vol. 10, No. 11 1163-1175
© 2001 Oxford University Press
Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down’s syndrome
1Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University and CREST (JST), Nishimachi 86, Yonago, Tottori 683-8503, Japan, 2Pharmaceutical Research Laboratory, KIRIN Brewery Co. Ltd, Miyahara-cho 3, Takasaki, Gunma 370-1295, Japan, 3Department of Pathology, Saga Medical School, Nabeshima 5, Saga 849-8501, Japan, 4First Department of Anatomy, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago, Tottori 683-8503, Japan, 5Mitsubishi Kasei Institute of Life Sciences, Minamiooya 11, Machida, Tokyo 194-8511, Japan and 6Department of Physiology, Johns Hopkins University School of Medicine, 725 North Wolf Street, Baltimore, MD 21205, USA
Received 5 January 2001; Revised and Accepted 19 March 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Trisomy 21 (Ts21) is the most common live-born human aneuploidy; it results in a constellation of features known as Down’s syndrome (DS). Ts21 is the most frequent cause of congenital heart defects and the leading genetic cause of mental retardation. To investigate the gene dosage effects of an extra copy of human chromosome 21 (Chr 21) on various phenotypes, we used microcell-mediated chromosome transfer to create embryonic stem (ES) cells containing Chr 21. ES cell lines retaining Chr 21 as an independent chromosome were used to produce chimeric mice with a substantial contribution from Chr 21-containing cells. Fluorescence in situ hybridization and PCR-based DNA analysis revealed that Chr 21 was substationally intact but had sustained a small deletion. The freely segregating Chr 21 was lost during development in some tissues, resulting in a panel of chimeric mice with various mosaicism as regards retention of the Chr 21. These chimeric mice showed a high correlation between retention of Chr 21 in the brain and impairment in learning or emotional behavior by open-field, contextual fear conditioning and forced swim tests. Hypoplastic thymus and cardiac defects, i.e. double outlet right ventricle and riding aorta, were observed in a considerable number of chimeric mouse fetuses with a high contribution of Chr 21. These chimeric mice mimic a wide variety of phenotypic traits of DS, revealing the utility of mice containing Chr 21 as unique models for DS and for the identification of genes responsible for DS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Trisomy 21 (Ts21) is the most frequent live-born aneuploidy in humans. This trisomy produces Down’s syndrome (DS), which includes a variety of developmental anomalies including facial dysmorphology, congenital defects of heart and gut, infertility, immunodeficiencies and an increased incidence of leukemia (1). A major goal of DS research is to correlate dosage imbalance of specific genes from chromosome 21 (Chr 21) with different clinical aspects of the syndrome. This goal is facilitated by the recent report of the sequence of 99.7% of the long arm of Chr 21 (33.46 Mb) (2). A gene catalog of expressed sequences was developed from the initial annotation, including 127 known genes and 98 predicted genes. The Chr 21 gene catalog facilitates research into the mechanisms underlying features of DS. Comparison of the phenotype and genotype of individuals who are partially trisomic for Chr 21 has been used to localize genes whose dosage imbalance contributes to these features (3–5). However, the number of these individuals is limited and the presentation of DS, even with full Ts21, is highly variable, limiting the resolution of phenotype maps. Further, many aspects of the DS phenotype are in place at birth. Systematic studies of prenatal development cannot be carried out in humans (6).
Mouse models have been used to study phenotype–genotype correlations in DS (7). Transgenic approaches range from single gene transgenics to creation of ‘in vivo’ libraries in mice (8), in which overlapping or contiguous yeast artificial chromosomes (YACs) spanning 2 Mb Chr 21q22.2 were introduced into the mouse genome. One of the four YACs, containing the Dyrk1a (Minibrain) gene, was implicated in causing subtle behavioral defects when present in three copies. Mice with segmental trisomy for the distal end of mouse chromosome 16, which corresponds genetically to most of Chr 21, provide another genetic model for DS (9). Two mouse strains with segmental trisomy 16, Ts65Dn and Ts1Cje, carry an extra copy of the segment of mouse Chr 16 extending from Gabpa to Mx1 (
16 Mb) and Sod1 to Mx1 (10 Mb), respectively (9,10). Both strains have deficits in learning and memory. Ts65Dn mice show precise parallels to anomalies of the craniofacial skeleton (11) and the cerebellum (12) that are seen in DS. However, the heart defect characteristic of DS has not been observed in live-born Ts65Dn or Ts1Cje mice, nor do these mice show the histopathology of early-onset Alzheimer’s disease.
One limitation of partial trisomy 16 mouse models is that they are trisomic for only a subset of the genes on human Chr 21. Chromosome engineering techniques can be used to make precise deletions, insertions or translocations of targeted regions (13). Another approach is to produce mice containing an additional, entire or partial human Chr 21 using microcell-mediated chromosome transfer (MMCT) (14). We used MMCT to construct libraries of mouse A9 cells containing single human chromosomes derived from normal human fibroblasts (15–17). Transfer of human chromosomes or chromosome fragments (hCFs) from these libraries to rodent cells exhibiting various deficiencies has facilitated mapping and cloning of genes responsible for tumor suppression, cellular senescence and hereditary genetic disorders through functional complementation (18–22). This method has been used to generate transgenic animals harboring hCFs with functional human loci, or transchromosomic (Tc) mice (16,23). We have used transmittable hCFs of several megabases containing human immunoglobulin loci to create mice expressing diverse human antibody repertories (23). These results have suggested that human proteins are expressed in mice and interact appropriately with other proteins. Recently, Hernandez et al. (24) employed MMCT to generate chimeric mice Chr 21. However, specific phenotypic abnormalities and phenotype–genotype correlations have not been described for these mice (24).
We report here the generation via MMCT of chimeric mice containing partial Chr 21 as an independent chromosome. These mice demonstrate specific parallels to developmental anomalies seen in DS. Mice in which a high percentage of cells contained a Chr 21 demonstrated a wide range of behavioral abnormalities, indicating abnormal brain development and/or function. Of special note, we also observed hypoplastic thymus and cardiac defects in chimeric mouse fetuses, and the severity of these phenotypes was correlated with the percentage of cells containing a copy of Chr 21. This approach provides a mouse model for studying these critical aspects of the DS phenotype.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of embryonic stem (ES) cells containing Chr 21
We screened a library of mouse A9 cells containing single human chromosomes tagged with the neomycin-resistance gene (16) to identify A9 hybrid clones carrying an independent, intact human Chr 21. Microcells prepared from these donor A9 cells were fused with mouse TT2F ES cells (39,XO) (25), and hybrids containing Chr 21 were selected with G418. G418-resistant ES clones were obtained at a frequency of 5 x 10–6 per fused ES cell; 14 clones were obtained from three independent experiments. The transferred Chr 21 in each line was characterized by PCR and fluorescence in situ hybridization (FISH) analyses. FISH analysis with human COT-1 DNA probe revealed that the transferred Chr 21 was retained as an independent chromosome in each clone (Fig. 1A). PCR analyses with eight Chr 21-specific primers revealed that seven of 14 clones contained all the markers examined, while the other seven clones exhibited various deletions, resulting in formation of a panel of ES cells containing different regions of Chr 21 (Fig. 1B).
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Production of chimeric mice
Three representative clones, ES(#21)-7, -10 and -11, were chosen from the panel of ES(#21) cells and used to produce viable chimeric mice. The ES(#21) cells were injected into 8-cell stage embryos of albino MCH(ICR) mice and the embryos were transplanted into the uteri or oviducts of pseudopregnant mice. Viable chimeric mice with varying levels of contribution of the ES cells were obtained (Table 1). Among transplanted embryos, 1.7% (3/174) to 10.9% (20/183) produced live-born pups that survived >4 weeks, and 3.4% (9/266) to 9.3% (17/183) resulted in pre- and postnatal death. The latter is likely to be an underestimate, because some dead pups were likely to have been cannibalized before they could be ascertained. Based on coat color, ES(#21)-10 and -11 clones had an ability to contribute to >90% in an albino MCH(ICR) background (Fig. 2A). ES(#21)-7 produced only low percentage chimeras (<50%) and these were not analyzed further. High level chimeras were produced more efficiently from clone ES(#21)-10 than from ES(#21)-11.
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Retention of Chr 21 in chimeric mice
The amount of Chr 21 information in each chimeric mouse was determined using PCR analysis with 13 Chr 21 markers (Fig. 2B). All of the 11 markers retained in the ES(#21)-10 cell line were present in the ES(#21)-10 chimeras. In contrast, all three of the high percentage chimeras produced with ES(#21)-11 were deleted for SIM2 and PWP2, even though the parental cell line retained all 13 markers by PCR. This ES cell line may represent a mixed population of cells, some of which contain an intact Chr 21 and others, the deleted Chr 21. Unfortunately, the three ES(#21)-11 chimeras died in a laboratory accident. Thus, only chimeras from clone ES(#21)-10 live were examined further.
Coat color chimerism is one reflection of the contribution of ES cells, but retention of Chr 21 in ES-derived cells is not necessarily identical to chimerism. Mitotic errors might result in the loss of all or part of the freely segregating human chromosome. At another level, cells with the extra chromosome may be at a selective advantage or disadvantage in different tissues (26). Therefore, in situ analysis was used to determine the retention rate of Chr 21 in cells from brain, heart, liver, spleen, bone-marrow and in cultured tail fibroblasts of adult chimeric mice made from the ES(#21)-10 cells (Fig. 2C and D). A human COT-1 probe detected Chr 21 as an independent extra copy in tail fibroblasts, indicating that no recombination or rearrangement had taken place between human and mouse chromosomes (Fig. 2C). The retention rate of Chr 21 in different tissues of chimeric mice is summarized in Figure 2E. Retention rate varied considerably, even among different mice and among different tissues of the same mouse. Notably, the retention rate of Chr 21 was relatively high in brain and low in heart compared with other tissues tested.
We also examined the retention of Chr 21 in cortex, cerebellum and hippocampus from five mice showing high chimerism and which were used for behavioral analysis (Fig. 2F). No significant difference in retention was found between different brain regions in an individual mouse. The contribution of the ES cells judged from coat color and retention of the Chr 21 in brain were similar in these mice.
Transcripts of Chr 21 in chimeric mice
To determine whether human genes on the transferred Chr 21 were transcribed appropriately, total RNA isolated from tissues of chimeric mice was analyzed by RT–PCR using specific primers for human expressed sequence tags (ESTs). Results from chimeric mouse C10-2 are representative (Fig. 3). The brain-specific polypeptide, PEP19 (PCP4), was detected only in the brain (27), while the transcript for the ubiquitously expressed human G protein coupled inward rectifier potassium channel 2 (GIRK2) was detected in all the tissues tested (28). The expression pattern of SIM2 in the chimeric mouse mimicked the expression in the human brain, lung, heart, liver, kidney and muscle (29). At least some human genes on the transferred Chr 21 were expressed under appropriate tissue-specific transcriptional regulation in chimeric mice despite the human origin of the chromosome and its passage in a differentiated fibroblast cell line. This is consistent with our previous studies in chimeras with chromosomes 2, 14 or 22 (16).
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Germline transmission
Nine female chimeric mice derived from ES(#21)-10 which showed a high percentage contribution of ES cells were mated with albino MCH(ICR) male mice to test for germline transmission of Chr 21 (30). Two chimeric mice produced six offspring with the dominant agouti coat color, indicating that they originated from ES-derived germ cells, while three chimeric mice produced only albino offspring and the remaining four chimeric mice produced no offspring. Retention of Chr 21 in agouti offspring was tested by Alu-PCR in genomic DNA from tail biopsy (data not shown). Two of the six agouti pups retained human DNA. However, detailed PCR analysis showed that only a limited portion of Chr 21, including CBR1, SIM2 and HLCS markers, was transmitted to the offspring (data not shown). FISH analysis using human COT-1 DNA showed that despite the loss of all markers proximal to CBR1, the transmitted Chr 21 fragment was maintained as a freely segregating chromosome of
5 Mb, implying that the centromere was retained (data not shown). These Tc offspring were not examined further.
Learning and behavior
Chimeric mice that were made from ES(#21)-10 cells and had >90% ES contribution to coat color were subjected to a battery of learning and behavioral tasks. Spontaneous motor activity in a novel environment was measured using an open-field box. ES(#21)-10 chimeras showed less activity in exploratory behavior than control mice (Fig. 4A–C). Two-way analysis of variance (ANOVA) revealed significant differences between ES(#21)-10 chimeras and control mice in distance traveled [F(1,96) = 28.64, P < 0.0001], rearing behavior [F(1,96) = 53.69, P < 0.0001] and time spent in locomotion [F(1,96) = 65.61, P < 0.0001]. There was no significant difference in speed of movement [F(1,96) = 3.922, P = 0.051] (data not shown). These data demonstrated that the ES(#21)-10 chimeras were hypoactive or hypokinetic in exploratory behavior compared with the control mice. Further, there was a significant correlation between the retention rate of Chr 21 and a behavioral decline in all three activities, i.e. a higher percentage of chimerism in the brain resulted in more anomalous behavior (Fig. 4D–F).
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The contextual fear conditioning task requires the integrated neural circuit of the hippocampus and amygdala. For the training trial, mice were placed in a conditioning chamber where they received three sequential foot shocks. ES(#21)-10 chimeras and control mice displayed a gradual and comparable increase in freezing during the training trial (data not shown), demonstrating that the ES(#21)-10 chimeras did not have a performance deficit. However, when returned to the conditioning chamber 24 h after the training session, ES(#21)-10 chimeras showed a less conditioned freezing response than control mice (Fig. 5A). Two-way ANOVA on the mean percentage of freezing per min confirmed a significant difference between groups [F(1,42) = 52.32, P < 0.0001] and no significant difference in the time course [F(5,42) = 1.64, P = 0.17] (Fig. 5A). Further, the percentage of freezing in ES(#21)-10 chimeras and control mice correlated significantly with the contribution of Chr 21-containing cells in the brain (r = 0.849, P < 0.005) (Fig. 5B). These results revealed that the ES(#21)-10 chimeras had impairment in associative learning of fear to contextual stimulus.
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We examined two behaviors which might affect performance in fear conditioning tests, and found no difference between ES(#21)-10 chimeras and control mice. First, the light–dark choice test is a measure of anxiety or fear-related emotion in rodents (31). As shown in Fig. 5C, there was no significant difference in the percentage of time spent in the light compartment during a 30 min test period between ES(#21)-10 chimeras and control mice [F(1,96) = 0.004, P = 0.953; two-way ANOVA], although the ES(#21)-10 chimeras showed a stronger preference for the dark compartment than did control mice during the first 5 min of the test. Secondly, because a change in sensitivity to pain might affect performance in fear conditioning analysis (32), nociceptive reactions to foot shock were evaluated. We measured the minimal level of current required to elicit vocalization and jumping after contextual fear conditioning. There was no difference in the mean threshold for vocalization (P = 0.472, Student’s t-test) and jumping (P = 0.951) between ES(#21)-10 chimeras and control mice (Fig. 5D). These data demonstrated that pain sensitivity in the ES(#21)-10 chimeras was normal, which suggested that the ES(#21)-10 chimeras had impairment in associative learning of fear to contexual stimulus.
The forced swim test has been widely used for screening of antidepressant drugs (33). Mice that spend a longer time immobile (floating) in an inescapable and stressful situation are considered to reflect a ‘depressive-like behavior’ or passive coping strategy. We examined the stress response related with emotionality in ES(#21)-10 chimeras. On the first day, mice were placed in a cylindrical tank and forced to swim (Fig. 5E). The ES(#21)-10 chimeras exhibited immobility for a significantly longer time than the control mice. Two-way ANOVA on duration of immobility revealed a significant difference between groups [F(1,125) = 22.28, P < 0.0001] and in a time-course [F(4,125) = 11.33, P < 0.0001]. On the next day, ES(#21)-10 chimeras exhibited an increase in the duration of immobility during the first 3 min of the test compared with that in the first trial, while the control mice showed a slight decrease in immobility over the 15 min test compared with that in the first trial (Fig. 5F). Two-way ANOVA confirmed a significant difference in duration of immobility between groups [F(1,125) = 53.61, P < 0.0001] and a significant difference in the time-course [F(4,125) = 2.98, P < 0.05]. In contrast to previous tests, there were no significant correlations between the contribution of Chr 21 and duration of immobility in the first (r = 0.515, P = 0.16) and second trial (r = 0.639, P = 0.06).
Developmental anomalies in chimeric mouse fetuses
Live-born chimeras were produced from the ES(#21)-11 cell line, which retained an intact Chr 21 (Table 1). Since 50% or more of human conceptuses with Ts21 do not survive until birth (1) the persistence of an entire copy of Chr 21 might be supposed to disturb gestation in the mouse as well. The development of 34 prenatal fetuses was examined at 18 days post-coitum. Retention rate of Chr 21 was determined in tail fibroblasts of 21 chimeras with pigmented eyes. Seven of the 21 chimeric fetuses (33.3%) showed some degree of developmental retardation, which tended to be more pronounced in fetuses with a Chr 21 retention rate >78% (Table 2). Hypoplastic thymus was observed in six fetuses, five of which also had conotruncal malformations of the heart (see below). Thymic hypoplasia occurred more frequently in fetuses with Chr 21 retention >92% and did not correlate with body size. Thirteen control mice had no gross abnormality. No chimeric fetuses had remarkable alterations of the head and spine, nor of abdominal organs including liver, spleen, kidney and ureter, and all ES(#21)-11 chimeric fetuses had a patent anus.
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Cardiovascular anomalies in ES(#21)-11 chimeric fetuses
Conotruncal malformations were observed in seven ES(#21)-11 chimeric fetuses (33%) and four of these displayed hypoplastic pulmonary artery (Table 2). However, there was no case that showed hypoplastic ascending aorta and coarctation of the aorta. Double outlet right ventricle (DORV), in which the ascending aorta and main pulmonary artery originate from the right ventricle with a ventricular septal defect (Fig. 6A), occurred in six cases. There was one case of riding aorta (F21) in which the ascending aorta overrode the ventricular septal defect.
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Only the most severely affected fetus, F20, displayed remarkable pathology of the aorta and the pulmonary semilunar valves. F20 had a large ascending aorta originating from the right ventricle. There was a rudimentary main pulmonary artery extending to the ductus arteriosus on the left side of the aorta (Fig. 6B). Further observation revealed a thin main pulmonary artery that originated from the right ventricle without a pulmonary valve (Fig. 6C), and a narrow infundibulum. This was diagnosed as DORV.
Atrioventricular (AV) canal malformations were observed in 10 ES(#21)-11 chimeric fetuses (48%). Bilateral hypoplastic AV canal, including one case of mitral atresia (F17), was observed in six fetuses and hypoplastic left AV canal was exhibited in four chimeric fetuses, respectively. AV valves were mostly dysplastic and sometimes buried in the ventricular wall. AV septal defects, which are frequently observed in DS patients, were not observed in the chimeric mice. Myocardial layers of heart ventricles showed characteristic phenotypes among chimeric fetuses. A fetus with Chr 21 retention <10% had mostly normal development of ventricles (Fig. 6D and E), while the myocardial layers in fetuses with Chr 21 retention >68% showed increasing structural change with increased Chr 21 retention. These fetuses were characterized by ventricles filled with myocardial cells with trabecular patterns (Fig. 6F). Ventricles were sometimes occupied by more compact myocardial cells, obscuring the ventricular spaces.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transfer of human Chr 21 into mice has specific advantages over transgenic technologies in the study of DS. Although it may be an alternate method to generate mice with all the mouse genomic equivalent of human Chr 21, our method is much easier because mouse genes equivalent to human genes were distributed to several different chromosomes in mouse. Large segments of genomic DNA, the size of which are far beyond the maximum capacity of conventional cloning vectors such as YACs, can be transferred. This provides the complete, identical set of genes that are in dosage imbalance in humans with Ts21. Further, these genes are introduced in the context of their native cis-acting regulatory elements and chromatin structure. This should maximize temporal and tissue-specific gene expression and function under physiologically appropriate conditions, a situation that is difficult to mimic using conventional transgenes and expression vectors. Any effects on cell metabolism and division resulting from the physical presence of an extra independent chromosome will occur in these mice as well. We have some mice containing a different human chromosomal region, human chromosomes 2 and 14, and no gross anomalies were observed in these mice. These mice may not be the control mice. The exact control mice should be mice with different regions of human Chr 21, which can be made using chromosome engineering techniques.
Recently, Hernandez et al. (24) reported the production of chimeric mice containing Chr 21 fragments using irradiation MMCT (XMMCT). Chimeric mice contained transferred Chr 21 material, but genes on the transferred Chr 21 were not consistently expressed with appropriate tissue specificity in all chimeric mice. Initial characterization did not identify phenotypes that were consistent among mice sharing overlapping fragments. Our strategy differed from the previous one in several points. Firstly, the Chr 21 in our experiment was derived initially from normal human fibroblasts. Secondly, we assessed directly the retention of the transferred Chr 21 in multiple tissues of chimeric mice by FISH. This analysis demonstrated substantial differences in retention rate among different tissues of the same chimeric mouse. Coat color chimerism is not a precise indicator of the retention of the transferred Chr 21. Thirdly, the use of a non-male ES cell line, TT2F (karyotype 39,XO), allowed us to assess germline transmission of introduced human chromosomes. Males with DS and male Ts65Dn mice are sterile and, in fact, any trisomy virtually always results in male sterility (34).
Different Chr 21-containing ES lines showed substantial differences in the ability to form high percentage chimeras. In fact, none of the 16 chimeric mice with >50% Chr 21 retention contained an intact Chr 21, suggesting that the presence of an entire copy of the chromosome might be severely deleterious during mouse development, as is the case for Ts21 in human beings. Clone ES(#21)-10, which produced the highest frequency of high percentage chimeras, deleted a region which includes the ERG2 and ETS2 loci, suggesting that these or other genes from this region may be especially deleterious in combination with dosage imbalance of all the other Chr 21 genes.
ES(#21)-10 chimeras showed a variety of abnormalities in behavioral tests. A decline in exploratory behavior in an open-field test which we demonstrated in these chimeras is also seen in Ts1Cje and in TgN(sim2)1Sagu, which has three copies of the SIM2 gene (10,35). Like these, Ts65Dn and transgenic mice that overexpress the SIM2 gene show impairment in associative learning of fear to contextual stimulus (36,37). The increased immobility observed in ES(#21)-10 chimeras indicates that they exhibit a symptom of a depressive state which may reflect a defect in emotionality in a stressful situation. The hippocampus and cortex play a role in exploratory behavior and novelty-seeking (38), so these results imply that this trisomy affects development and/or function of these critical brain regions. It is notable that there was a significant correlation between these behavioral deficits, and the retention rate of Chr 21 in the hippocampus and cortex was significantly high. Since Baxter et al. (12) have recently shown cerebellar pathology, it is interesting to examine the presence of abnormalities in our mice and correlate with behavioral impairment.
A range of cardiovascular anomalies was seen in chimeric mice in which a high percentage of cells contained human Chr 21. These anomalies did not parallel precisely those seen most commonly in DS. For example, AV septal defect, a specific cardiovascular malformation (39) that comprises 10% of cardiovascular malformation in DS (40), was not observed in this study. However, the pattern of heart defects in chimeric mice, which frequently included DORV and riding aorta (seven cases, 33%), hypoplastic main pulmonary artery and tetralogy of Fallot, reflect maldevelopment of structures with a substantial input of neural crest. Defects in neural crest generation, migration and/or differentiation have been posited to play a central role in DS, since many structures are affected to which these cells make a significant contribution.
Hypoplastic AV canal seen in various chimeras may be developmentally related to the abnormal architecture of the ventricular myocardium, depending on the view that valvular leaflets have a muscular origin (41). Abnormal myocardial cells with a trabecular pattern were seen frequently in early embryonic hearts. Myocardial trabeculations, which are necessary for the blood supply in early embryonic hearts (42), are arranged and integrated in the process of ventricular development. This process would likely be impaired in the hearts of ES(#21)-11 chimeras. The fact that the presence of Chr 21 in mouse ES cells delays differentiation of cardiomyocytes in vitro (43) may be related to the impairment of ventricular development of ES(#21)-11 chimera hearts. The ventricular structure of human Ts21 fetuses does not usually show this kind of abnormality (39).
Considerable individual phenotypic variation was observed in prenatal ES(#21)-11 chimeric fetuses. This variation mimics the situation in DS, where the majority of the >80 diagnostic features are present in only a subset of individuals with Ts21. There may be several sources of this variation. The first is the degree to which the chimeras are populated with trisomic cells. In general, more abnormalities of greater severity were seen in fetuses with a greater portion of cells with Chr 21. However, even among fetuses with >80% Chr 21 contribution, growth retardation, hypoplastic thymus and cardiovascular malformations were not associated uniformly. A second source of variation is the different Chr 21 retention rate in different tissues. The retention rate of the introduced Chr 21 could not be tested in heart and thymus but was observed in tail fibroblast. Differences between individual fetuses could have contributed to inconsistency in the severity of the phenotypes observed. Cardiovascular malformations are observed in 40–50% of DS patients (44,45). Eleven cases (52.4%) of chimeric fetuses had cardiovascular malformations in the conotruncus and AV canal. The finding in ES(#21)-10 chimeras that the retention rate of Chr 21 was lowest in the heart among all the organs examined may explain the variable presence or absence of abnormality in the cardiovascular system of ES(#21)-11 chimeric fetuses.
Recently we reported that increased dosage of Chr 21 affects differentiation of ES cells in vitro (43). In that study, ES cells with Chr 21 exhibited a delay in the appearance of beating cardiomyocytes during differentiation, whereas differentiation into other cell lineages was not disrupted. These results suggest that delayed differentiation may have contributed to the disturbance of cardiogenesis in our chimeric mice. In this model, a higher contribution of trisomic cells in high retention rate chimeras would delay heart development to a degree that could not be compensated by cells from the euploid parent of the chimera. The occurrence of heart pathology in these mice provides a model system to investigate the cardiac defects of human DS, which is not possible in segmental trisomy 16 models.
We report effects on mouse development of dosage imbalance for most genes on human Chr 21. Introduction of human Chr 21 provides the most precise genetic system for assessing the dosage imbalance in Ts21. Despite the fact that virtually all chromosomal structural and regulatory proteins on this human Chr 21 derive from mouse, and that the proteins themselves represent human and not mouse versions of these gene products, several important aspects of DS are recapitulated in these mice, including heart defects that are not observed in any other genetic models. However, each chimeric mouse has a different pattern of trisomic and euploid cells, complicating analysis. We are developing mice that have Chr 21 and defined fragments of Chr 21 translocated to a mouse chromosome, using the Cre-loxP chromosome engineering techniques (46,47). These mice should allow stable germ-line transmission of the included human genetic information, circumventing the inherent difficulties in analysis of chimeras. ‘Gene knockouts’ of the included Chr 21 genes will provide a powerful system to identify those contributing to the central nervous system and heart pathology of DS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MMCT
The mouse ES line, TT2F (Lifetech Oriental), was maintained in an undifferentiated state by culture on G418-resistant primary embryonic fibroblasts (16). MMCT was performed as described previously (16). Briefly, mouse A9 cells containing a Chr 21 tagged with pSTneo were used as a donor source for MMCT into mouse ES cells. The hybrid cells were maintained in non-selective culture medium for 24 h and then selected in culture medium containing 300 µg/ml G418. We isolated G418-resistant clones between days 7 and 9 after applying selection and screened genomic DNA from these clones by PCR using Chr 21-specific marker analysis.
Genomic DNA analysis
PCR analyses were carried out using standard techniques. The following markers and primer pairs were used: D21S1904, 5'-ATGAGTTCAGTGTTTCATGGACACT-3' and 5'-AGCAAGATTACTTGTCTGGTTTCCC-3'; APP (GenBank accession no. SHGC-31514), 5'-CTGGGCAATAGAGCAAGACC-3' and 5'-ACCCATATTATCTATGGACAATTGA-3', amplifies a 115 bp fragment; D21S260, 5'-AGCTGTTCATGCTTCCATCT-3' and 5'-AGAGCCCAGAATATTGACCC-3', 270 bp; SOD1 (accession no. SHGC-6902), 5'-ATTCTGTGATCTCACTCTCAGG-3' and 5'-TCGCGACTAACAATCAAAGT-3', 133 bp; CBR1 (accession no. J04056), 5'-GATCCTCCTGAATGCCTG-3' and 5'-GTAAATGCCCTTTGGACC-3', 245 bp; ETS2 (accession no. SHGC-6939), 5'-TCGTGGACACACACAGACTA-3' and 5'-CTTTACAACGTCTCTTAGTCGG-3', 337 bp; D21S268, 5'-CAACAGAGTGAGACAGGCTC-3' and 5'-TTCCAGGAACCACTACACTG-3', 213 bp; COL18A1 (accession no. stSG1580), 5'-TTTATTTGCCTGTCTGAATTGG-3' and 5'-AAAGCAGCCACGAGGTGC-3', 227 bp; STCH (accession no. SHGC-10662), 5'-TTTTGTCTTAGGATTAGACGTGACC-3' and 5'-AGAACTGGGAAGTCTCATAACTGG-3', 215 bp; SIM2 (accession no. WI-22186), 5'-GGGCCTCATGGTAAGAGTCA-3' and 5'-GAAAAATGTCGGTGGTATCTCC-3', 250 bp; ERG (accession no. M21535), 5'-AATGGCGTCAGCCTCTCC-3' and 5'-CAGTTTGCCTTACGAGTGGTAGC-3', 254 bp; MX1 (accession no. WI-18875), 5'-TGGACTGACGACTTGAGTGC-3' and 5'-CTCATGTGCATCTGAGGGTG-3', 143 bp; PCP4 (accession no. WI-14954), 5'-GAATTCACTCATCGTAACTTCATTT-3' and 5'-CCTTGTAGGAAGGTATAGACAATGG-3', 126 bp; PWP2 (accession no. SHGC-33273), 5'-GATCTTGACCGGGAAAAGGG-3' and 5'-AACAAGTGGCAAAATGCATAC-3', 150 bp [GeneMap ’99 (National Center for Biotechnology Information); http://www.ncbi.nlm.nih.gov/genemap/]; and GIRK2 (GIRK2F, 5'-CCCAAAATACTACACATCC-3' and GIRK2R, 5'-GTTTGTCTTCAGCTCACC-3', 266 bp) (24). Amplifications were performed with an annealing temperature of 62°C for 30 cycles and the products were analyzed on a 3% agarose gel.
RT–PCR
Total RNA was prepared from brain, lung, heart, liver, kidney and skeletal muscle of control (F1; C57BL/6 x CBA) and chimeric mice using ISOGEN (Nippon gene). RNA was reverse transcribed with Superscript RTase (Gibco BRL) using random hexamers and first strand cDNA was used as a template for PCR. Human Multiple Tissue cDNA(MTC) Panel I (Clontech) was used for human cDNA control. Primer pairs for PCP4 and SIM2 were the same as genomic PCR and GIRK2 (GIRKRNA2F, 5'-TTCATCCCGTTGAACCAGACGG-3' and GIRKRNA2R, 5'-CCCATCCTCCAGGGTCAGGAC-3', amplifies a 281 bp fragment) (24). GAPDH was 5'-CCATCTTCCAGGAGCGAGA-3' and 5'-TGTCATACCAGGAAATGAGC-3', 722 bp. Amplifications were performed with an annealing temperature of 62°C for 30 cycles and the products were analyzed on a 3% agarose gel.
FISH analysis
Chromosome samples and FISH analysis were carried out by standard methods. Images were captured using a Nikon fluorescence microscope equipped with a photometric CCD camera and processed using the Cytovision Probe System (Applied Imaging). The probes were digoxigenin (Boehringer)-labeled human COT-1 DNA (BRL) and the pSTneo probe. Digoxigenin-labeled probes were detected with anti-digoxigenin-rhodamine (Boehringer). The chromosomes were counter-stained with DAPI (Sigma).
Chimera production
Chimera production was carried out as described previously (16). Briefly, ES cells were injected into 8-cell stage embryos derived from jcl: MCH(ICR) mice (Crea Japan), and transferred into pseudopregnant jcl: MCH(ICR) females.
Behavioral tests
Female mice (7–10 months of age) were used for behavioral analysis. Experiments were performed on five ES(#21)-10 chimeras and four C57BL/6 x CBA-F1 mice. The TT2F ES line was derived from the C57BL/6 x CBA-F1 strain and gives an agouti coat color in an albino MCH(ICR) background. The degree of chimerism, as determined by coat pigmentation, was 99% in chimeras C10-5, -6, -7 and -8 and 90% in C-9. The average retention of Chr 21 in three regions of the brain was determined after behavioral analysis was complete (text and Fig. 2F). The C-8 mouse showed only 2% chimerism in brain and was included in the control group. The mice were kept on a 12 h light–dark cycle with constant temperature (23 ± 1°C). Tests were always conducted between 13.00 and 18.00 h. One week before the beginning of behavioral tests, the mice were housed one per cage and handled once a day for 5 days. Behavioral tests were performed blind.
All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals and were approved by the Animal Care and Use Committee of the Mitsubishi Kasei Institute of Life Sciences. All efforts were made to minimize animal suffering and to reduce the number of animals used.
General activity
In the open-field test, spontaneous activity was measured in a novel environment using an open-field box (50 x 50 x 40 cm) placed in a sound-attenuating room by the method described previously (48). Two pairs of 24 x 24 array infrared photosensors were attached to the outer wall, equally spaced in lower and upper rows. The lower row of photocells measured locomotion activity and the upper row detected rearing behavior. A computer measured the distance of locomotion, the frequency of rearing, the time spent in movement (>1 cm/s) and the speed of movement. Each mouse remained in the apparatus for 30 min. Trials were duplicated at an interval of 2 days for each mouse to compensate for variations in spontaneous motor activity, which is dependent on the estrous cycle. Mean values of activity counts obtained from two trials were used as activity scores for each mouse.
Light–dark choice test
The apparatus consisted of two compartments and was placed in a darkened and sound-attenuating room. One compartment was a bright (250 lux) chamber (25 x 25 x 40 cm) illuminated by a white bulb (100 W) and the other was a dark (0.5 lux) chamber (25 x 25 x 40 cm). The two compartments were separated by a wall and connected by a small opening (8 x 16 cm). A mouse was placed in the center of the light chamber facing the openings and remained in the apparatus for 30 min. Movements of mice were detected by photosensors. The mouse was considered to have entered a new area when all four feet were in this area. The following behavioral measures were scored by a computer: the time spent in the light and dark compartments, the number of transitions between the two compartments and the latency of the initial movement from the light to the dark room.
Contextual fear conditioning
The conditioning chamber was an observation box (20 x 20 x 20 cm) made of clear and gray vinylchloride plates. The floor of the chamber consisted of 26 stainless steel rods through which foot shocks were delivered by a scrambled-foot shock generator (SGS 002, Muromachi). The chamber was placed in another sound-attenuating room. The chamber was cleaned with ethanol (99.5%) and dried with a hair drier to refresh the air before and after the occupancy of each mouse. A video camera placed in front of the chamber recorded the behavior of each mouse. On the training day, each mouse was placed in the chamber for 2.5 min and subsequently received three foot shocks (0.5 mA intensity, 1 s duration, 1 min interval). The mice were removed from the chamber 1 min after the last foot shock and returned to their home cages. Twenty-four hours after the training session, the mice were again placed in the chamber and tested for 6 min without any foot shocks. Freezing behavior was used to assess the degree of conditioned fear of the chamber. Freezing behavior was defined as the absence of visible movement, except for respiration. During the test period, a trained observer who was blind to the experimental conditions scored the tendency for the mouse to freeze by watching a TV monitor placed outside the sound-attenuated room. The observation was carried out using a time sampling procedure. Every 5 s, each mouse was judged as either freezing or active during the test. The time spent in freezing was calculated per min.
Electric shock sensitivity test
We measured the sensitivity of mice to foot shock, because foot shock sensitivity may affect freezing responses. In this test, each mouse was placed in the conditioning chamber and received 1 s shocks of increasing intensity. The interval between shocks was 10 s. The sequence of the current used was as follows: 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.8 mA. We determined the minimal level of current required to elicit the following stereotypical responses; vocalization and jumping. These experiments were performed blindly.
Forced swim test
On the first day of the test, each mouse was placed and kept in the water for 15 min. The test was carried out in a cylindrical plexiglass tank (25 cm high and 20 cm in diameter), filled with water (25 ± 1°C) up to a level of 15 cm. On the second day, 24 h later, the mouse was placed in the tank for 15 min. After each swim session, the mouse was dried gently with paper towel and returned to its home cage. These trials were videotaped for later analysis. The mouse’s movement during the swim test was measured using an infrared sensor system with a multi-Fresnel lens (CompACT FSS system, Muromachi) (49) which was placed 15 cm directly above the tank. The sensor monitors movement by detecting any object with a temperature 
5°C higher than background within the tank. Behavior in the tank was scored every 1 s as either swimming or immobility by a computer on the criteria of movement counts detected by the sensor. Immobility was defined as behavior of less than one movement count per second, which was about the same level that a trained observer judged as immobility while viewing the videotape. The time spent in immobility was calculated per min.
Data analysis
Experimental data were analyzed by two-way ANOVA and the Student’s t-test. Correlation coefficients between behavioral data and level of chimerism were calculated by Pearson’s product–moment correlation. Correlation magnitudes were compared between them by transforming the correlations to z values using the Fisher r to z transformation and then testing for significant differences. Values of P < 0.05 were considered as statistically significant. All values in the text and figure legends are expressed as mean ± SEM, and n is the number of mice tested. The C-8 mouse was included in the control group, because of its low contribution of Chr 21.
Phenotypic analysis of ES(#21)-11 chimera fetuses
Chimeric mouse fetuses produced with ES(#21)-11 were removed by sacrificing 34 dams on 18 days of pregnancy. The percentage of Chr 21 retained in tail fibroblasts of 21 chimeras with pigmented eyes was examined by FISH analysis of 50 mitotic spreads from tail biopsies, and was a function of the percentage of chimerism and retention of the introduced Chr 21. After fixation in 10% formalin solution buffered by phosphate, a total of 21 chimeric fetuses were microdissected under a stereomicroscope. Eight fetuses were chosen for histological examination of the cardiovascular system. Thirteen non-chimeric fetuses were examined as the control.
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ACKNOWLEDGEMENTS |
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The authors are deeply indebted to Mr Hiroyuki Ideguchi, Pathology Laboratory, Saga Medical School, for his kind technical assistance, Dr T.C. Schulz for comments on the manuscript, Dr Koutaro Inoue, Mitsubishi Kasei Institute of Life Sciences, for technical assistance and Drs T. Inoue-Nishida, K. Mitsuya and Y. Shirayoshi, Tottori University, for valuable comments. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, by CREST of the Japan Science and Technology Corporation (M.O.), by Public Health Service awards HD38384 and HDS24605 (R.R.) and from the Research Project of the Drug Safety Agency (M.O.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 859 34 8260; Fax: +81 859 34 8134; Email: oshimura@grape.med.tottori-u.ac.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Epstein, C.J. (1986) Consequences of Chromosome Imbalance: Principals, Mechanisms and Models. Cambridge University Press, New York, NY.
2 Hattori, M., Fujiyama, A., Taylor, T.D., Watanabe, H., Yada, T., Park, H.S., Toyoda, A., Ishii, K., Totoki, Y., Choi, D.K. et al. (2000) DNA sequence of human chromosome 21. Nature, 405, 311–319.[Medline]
3 McCormick, M.K., Schinzel, A., Petersen, M.B., Stetten, G., Driscoll, D.J., Cantu, E.S., Tranebjaerg, L., Mikkelsen, M., Watkins, P.C. and Antonarakis, S.E. (1989) Molecular genetic approach to the characterization of the ‘Down syndrome region’ of chromosome 21. Genomics, 5, 325–331.[Web of Science][Medline]
4 Korenberg, J.R., Chen, X.N., Schipper, R., Sun, Z., Gonsky. R., Gerwehr, S., Carpenter, N., Daumer, C., Dignan, P., Disteche, C. et al. (1994) Down syndrome phenotypes: the consequences of chromosomal imbalance. Proc. Natl Acad. Sci. USA, 24, 4997–5001.
5 Rahmani, Z., Blouin, J.L., Creau-Goldberg, N., Watkins, P.C., Mattei, J.F., Poissonnier, M., Prieur, M., Chettouh, Z., Nicole, A., Aurias, A. et al. (1989) Critical role of the D21S55 region on chromosome 21 in the pathogenesis of Down syndrome. Proc. Natl Acad. Sci. USA, 86, 5958–5962.
6 Reeves, R.H., Baxter, L.L. and Richtsmeier, J.T. (2001) Too much of a good thing: mechanisms of gene action in Down syndrome. Trends Genet., 17, 79–84.[Web of Science][Medline]
7 Kola, I. and Hertzog, P.J. (1997) Animal models in the study of the biological function of genes on human chromosome 21 and their role in the pathophysiology of Down syndrome. Hum. Mol. Genet., 6, 1713–1727.
8 Smith, D.J., Stevens, M.E., Sudanagunta, S.P., Bronson, R.T., Makhinson, M., Watabe, A.M., O’Dell, T.J., Fung, J., Weier, H.U., Cheng, J.F. et al. (1997) Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome. Nat. Genet., 16, 28–36.[Web of Science][Medline]
9 Reeves, R.H., Irving, N.G., Moran, T.H., Wohn, A., Kitt, C., Sisodia, S.S., Schmidt, C., Bronson, R.T. and Davisson, M.T. (1995) A mouse model for Down syndrome exhibits learning and behaviour deficits. Nat. Genet., 11, 177–184.[Web of Science][Medline]
10 Sago, H., Carlson, E.J., Smith, D.J., Kilbridge, J., Rubin, E.M., Mobley, W.C., Epstein, C.J. and Huang, T.T. (1998) Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc. Natl Acad. Sci. USA, 26, 6256–6261.
11 Richtsmeier, J.T., Baxter, L.L. and Reeves, R.H. (2000) Parallels of craniofacial maldevelopment in Down Syndrome and Ts65Dn mice. Dev. Dyn., 217, 137–145.[Web of Science][Medline]
12 Baxter, L.L., Moran, T.H., Richtsmeier, J.T., Troncoso, J. and Reeves, R.H. (2000) Discovery and genetic localization of Down syndrome cerebellar phenotypes using the Ts65Dn mouse. Hum. Mol. Genet., 22, 195–202.
13 Hernandez, D. and Fisher, E.M. (1999) Mouse autosomal trisomy: two’s company, three’s a crowd. Trends Genet., 15, 241–247.[Web of Science][Medline]
14 Fournier, R.E.K. and Ruddle, F.H. (1977) Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells. Proc. Natl Acad. Sci. USA, 74, 319–323.
15 Koi, M., Shimizu, M., Morita, H., Yamada, H. and Oshimura, M. (1989) Construction of mouse A9 clones containing a single human chromosome tagged with neomycin-resistance gene via microcell fusion. Jpn. J. Cancer Res., 80, 413–418.[Web of Science][Medline]
16 Tomizuka, K., Yoshida, H., Uejima, H., Kugoh, H., Sato, K., Ohguma, A., Hayasaka, M., Hanaoka, K., Oshimura, M. and Ishida, I. (1997) Functional expression and germline transmission of a human chromosome fragment in chimaeric mice. Nat. Genet., 16, 133–143.[Web of Science][Medline]
17 Kugoh, H., Mitsuya, K., Meguro, M., Shigenami, K., Schulz, T.C. and Oshimura, M. (1999) Mouse A9 cells containing single human chromosomes for analysis of genomic imprinting. DNA Res., 30, 165–172.
18 Seya, T., Okada, M., Hara, T., Matsumoto, M., Miyagawa, S. and Oshimura, M. (1991) Human complement regulatory proteins expressed on mouse A9 cells containing a human chromosome 1. Immunology, 74, 719–724.[Web of Science][Medline]
19 Kurimasa, A., Ohno, K. and Oshimura, M. (1993) Restoration of the cholesterol metabolism in 3T3 cell lines derived from the sphingomyelinosis mouse (spm/spm) by transfer of a human chromosome 18. Hum. Genet., 92, 157–162.[Web of Science][Medline]
20 Kurimasa, A., Nagata, Y., Shimizu, M., Emi, M., Nakamura, Y. and Oshimura, M. (1994) A human gene that restores the DNA-repair defect in SCID mice is located on 8p11.1
q11.1. Hum. Genet., 93, 21–26.[Web of Science][Medline]
21 Zenklusen, J.C., Oshimura, M., Barrett, J.C. and Conti, C.J. (1995) Human chromosome 11 inhibits tumorigenicity of a murine squamous cell carcinoma cell line. Genes Chromosomes Cancer, 13, 47–53.[Web of Science][Medline]
22 Nihei, N., Ichikawa, T., Kawana, Y., Kuramochi, H., Kugo, H., Oshimura, M., Killary, A.M., Rinker-Schaeffer, C.W., Barrett, J.C., Isaacs, J.T. et al. (1995) Localization of metastasis suppressor gene(s) for rat prostatic cancer to the long arm of human chromosome 10. Genes Chromosomes Cancer, 14, 112–119.[Web of Science][Medline]
23 Tomizuka, K., Shinohara, T., Yoshimda, H., Uejima, H., Ohguma, A., Tanaka, S., Sato, K., Oshimura, M. and Ishida, I. (2000) Double trans-chromosomic mice: Maintenance of two individual human chromosome fragments containing immunoglobulin heavy and 
loci and expression of fully human antibodies. Proc. Natl Acad. Sci. USA, 97, 722–727.
24 Hernandez, D., Mee, P.J., Martin, J.E., Tybulewicz, V.L. and Fisher, E.M. (1999) Transchromosomal mouse embryonic stem cell lines and chimeric mice that contain freely segregating segments of human chromosome 21. Hum. Mol. Genet., 8, 923–933.
25 Uchida, M., Tokunaga, T., Aizawa, S., Niwa, K. and Imai, H. (1995) Isolation and nature of mouse embryonic stem cell line efficiently producing female germ-line chimeras. Anim. Sci. Technol. (Jpn.), 66, 361–367.
26 Shinohara, T., Tomizuka, K., Takehara, S., Yamauchi, K., Katoh, M., Ohguma, A., Ishida, I. and Oshimura, M. (2000) Stability of transferred human chromosome fragments in cultured cells and in mice. Chromosome Res., 8, 713–725.[Web of Science][Medline]
27 Ziai, M.R., Sangameswaran, L., Hempstead, J.L., Danho, W. and Morgan, J.I. (1988) An immunochemical analysis of the distribution of a brain-specific polypeptide, PEP-19. J. Neurochem., 51, 1771–1776.[Web of Science][Medline]
28 Mjaatvedt, A.E., Cabin, D.E., Cole, S.E., Long, L.J., Breitwieser, G.E. and Reeves, R.H. (1995) Assessment of a mutation in the H5 domain of Girk2 as a candidate for the weaver mutation. Genome Res., 5, 453–463.
29 Chrast, R., Scott, H.S., Chen, H., Kudoh, J., Rossier, C., Minoshima, S., Wang, Y., Shimizu. N. and Antonarakis, S.E. (1997) Cloning of two human homologs of the Drosophila single-minded gene SIM1 on chromosome 6q and SIM2 on 21q within the Down syndrome chromosomal region. Genome Res., 7, 615–624.
30 Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994) Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, NY.
31 Costall, B., Jones, B.J., Kelly, M.E., Naylor, R.J. and Tomkins, D.M. (1989) Exploration of mice in a black and white test box: validation as a model of anxiety. Pharmacol. Biochem. Behav., 32, 777–785.[Web of Science][Medline]
32 Martinez-Cue, C., Baamonde, C., Lumbreras, M.A., Vallina, I.F., Dierssen, M. and Florez, J. (1999) A murine model for Down syndrome shows reduced responsiveness to pain. Neuroreport, 10, 1119–1122.[Web of Science][Medline]
33 Porsolt, R.D., Anton, G., Blavet, N. and Jalfre, M. (1978) Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur. J. Pharmacol., 47, 379–391.[Web of Science][Medline]
34 Davisson, M.T., Schmidt, C., Reeves, R.H., Irving, N.G., Akeson, E.C., Harris, B.S. and Bronson, R.T. (1993) Segmental trisomy as a mouse model for Down syndrome. Prog. Clin. Biol. Res., 384, 117–133.[Medline]
35 Chrast, R., Scott, H.S., Madani, R., Huber, L., Wolfer, D.P., Prinz, M., Aguzzi, A., Lipp, H.P. and Antonarakis, S.E. (2000) Mice trisomic for a bacterial artificial chromosome with the single-minded 2 gene (Sim2) show phenotypes similar to some of those present in the partial trisomy 16 mouse models of Down syndrome. Hum. Mol. Genet., 22, 1853–1864.
36 Ema, M., Ikegami, S., Hosoya, T., Mimura, J., Ohtani, H., Nakao, K., Inokuchi, K., Katsuki, M. and Fujii-Kuriyama, Y. (1999) Mild impairment of learning and memory in mice overexpressing the mSim2 gene located on chromosome 16: an animal model of Down’s syndrome. Hum. Mol. Genet., 8, 1409–1415.
37 Coussons-Read, M.E. and Crnic, L.S. (1996) Behavioral assessment of the Ts65Dn mouse, a model for Down syndrome: altered behavior in the elevated plus maze and open field. Behav. Genet., 26, 7–13.[Web of Science][Medline]
38 Sandin, J., Georgieva, J., Schott, P.A., Ogren, S.O. and Terenius, L. (1997) Nociceptin/orphanin FQ microinjected into hippocampus impairs spatial learning in rats. Eur. J. Neurosci., 9, 194–197.[Web of Science][Medline]
39 Miyabara, S., Ando, M., Suzumori, K., Nishibatake, M., Saito, N., Sugihara, H. and Ikenoue, T. (2000) Incidence and types of congenital cardiovascular malformations in Japanese trisomy 21 fetuses around 20 weeks. Cong. Anom., 40, 117–122.
40 Stoll, C., Alembik, Y., Dott, B. and Roth, M.P. (1990) Epidemiology of Down syndrome in 118 265 consecutive births. Am. J. Med. Genet., 7, 79–83.
41 Wenink, A.C.G. and Zevallos, J.C. (1988) Developmental aspects of atrioventricular septal defect. Int. J. Cardiol., 18, 65–78.[Web of Science][Medline]
42 Tokuyasu, K.T. (1990) Co-development of embryonic myocardium and myocardial circulation. In Clark, E.B. and Takao, A. (eds), Developmental Cardiology: Morphology and Function, Futura Pub., Mount Kisco, NY, pp. 205–218.
43 Inoue, T., Shinohara, T., Takehara, S., Inoue, J., Kamino, H., Kugoh, H. and Oshimura, M. (2000) Specific impairment of cardiogenesis in mouse ES cells containing a human chromosome 21. Biochem. Biophys. Res. Commun., 24, 219–224.
44 Rowe, R.D. and Uchida, I.A. (1961) Cardiac malformation in mongolism: A prospective study of 184 mongoloid children. Am. J. Med., 31, 726–735.[Web of Science][Medline]
45 Khoury, M.J. and Erickson, J.D. (1992) Improved ascertainment of cardiovascular malformations in infants with Down’s syndrome, Atlanta, 1968 through 1989. Am. J. Epidemiol., 136, 1457–1464.
46 Ramirez-Solis, R., Liu, P. and Bradley, A. (1995) Chromosome engineering in mice. Nature, 378, 720–724.[Medline]
47 Kuroiwa, Y., Tomizuka, K., Shinohara, T., Kazuki, Y., Yoshida, H., Ohguma, A., Yamamoto, T., Tanaka, S., Oshimura, M. and Ishida, I. (2000) Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts. Nat. Biotechnol., 18, 1086–1090.[Web of Science][Medline]
48 Ikegami, S., Harada, A. and Hirokawa, N. (2000) Muscle weakness, hyperactivity, and impairment in fear conditioning in tau-deficient mice. Neurosci. Lett., 279, 129–132.[Web of Science][Medline]
49 Masuo, Y., Matsumoto, Y., Morita, S. and Noguchi, J. (1997) A novel method for counting spontaneous motor activity in the rat. Brain Res. Brain Res. Protoc., 1, 321–326.[Medline]
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