唐氏综合征小鼠模型的遗传背景和应用

唐氏综合征(Down syndrome, DS)是最常见的常染色体异常疾病,由人类21号染色体(human chromosome 21, Hsa21)的重复引起。由于Hsa21的直系同源基因分散于小鼠16、17和10号染色体上,所以用小鼠模拟人类唐氏综合征并不容易。早期的Ts65Dn小鼠虽然具有DS表型特征,但其重复片段由电离辐射产生,未包含所有Hsa21直系同源基因。2004年,Cre/LoxP重组酶系统介导的染色体编辑技术在Ts1Rhr小鼠中的成功应用,解决了特定片段重复化的难题,使DS小鼠模型在基因重复和表型模拟方面实现了精准化。本文从同源基因重复和DS表型模拟两方面简要介绍了不同时期DS小鼠模型的优势和局限,为科研人员在DS研究中对不同小鼠模型的选用提供了参考。     

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.     

Mouse models of Down syndrome: gene content and consequences

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), is challenging to model in mice. Not only is it a contiguous gene syndrome spanning 35 Mb of the long arm of Hsa21, but orthologs of Hsa21 genes map to segments of three mouse chromosomes, Mmu16, Mmu17, and Mmu10. The Ts65Dn was the first viable segmental trisomy mouse model for DS; it is a partial trisomy currently popular in preclinical evaluations of drugs for cognition in DS. Limitations of the Ts65Dn are as follows: (i) it is trisomic for 125 human protein-coding orthologs, but only 90 of these are Hsa21 orthologs and (ii) it lacks trisomy for ~75 Hsa21 orthologs. In recent years, several additional mouse models of DS have been generated, each trisomic for a different subset of Hsa21 genes or their orthologs. To best exploit these models and interpret the results obtained with them, prior to proposing clinical trials, an understanding of their trisomic gene content, relative to full trisomy 21, is necessary. Here we first review the functional information on Hsa21 protein-coding genes and the more recent annotation of a large number of functional RNA genes. We then discuss the conservation and genomic distribution of Hsa21 orthologs in the mouse genome and the distribution of mouse-specific genes. Lastly, we consider the strengths and weaknesses of mouse models of DS based on the number and nature of the Hsa21 orthologs that are, and are not, trisomic in each, and discuss their validity for use in preclinical evaluations of drug responses.     

The power of comparative and developmental studies for mouse models of Down syndrome

Since the genetic basis for Down syndrome (DS) was described, understanding the causative relationship between genes at dosage imbalance and phenotypes associated with DS has been a principal goal of researchers studying trisomy 21 (Ts21). Though inferences to the gene-phenotype relationship in humans have been made, evidence linking a specific gene or region to a particular congenital phenotype has been limited. To further understand the genetic basis for DS phenotypes, mouse models with three copies of human chromosome 21 (Hsa21) orthologs have been developed. Mouse models offer access to every tissue at each stage of development, opportunity to manipulate genetic content, and ability to precisely quantify phenotypes. Numerous approaches to recreate trisomic composition and analyze phenotypes similar to DS have resulted in diverse trisomic mouse models. A murine intraspecies comparative analysis of different genetic models of Ts21 and specific DS phenotypes reveals the complexity of trisomy and important considerations to understand the etiology of and strategies for amelioration or prevention of trisomic phenotypes. By analyzing individual phenotypes in different mouse models throughout development, such as neurologic, craniofacial, and cardiovascular abnormalities, greater insight into the gene-phenotype relationship has been demonstrated. In this review we discuss how phenotype-based comparisons between DS mouse models have been useful in analyzing the relationship of trisomy and DS phenotypes.     

Opposite Phenotypes of Muscle Strength and Locomotor Function in Mouse Models of Partial Trisomy and Monosomy 21 for the Proximal Hspa13-App Region

The trisomy of human chromosome 21 (Hsa21), which causes Down syndrome (DS), is the most common viable human aneuploidy. In contrast to trisomy, the complete monosomy (M21) of Hsa21 is lethal, and only partial monosomy or mosaic monosomy of Hsa21 is seen. Both conditions lead to variable physiological abnormalities with constant intellectual disability, locomotor deficits, and altered muscle tone. To search for dosage-sensitive genes involved in DS and M21 phenotypes, we created two new mouse models: the Ts3Yah carrying a tandem duplication and the Ms3Yah carrying a deletion of the Hspa13-App interval syntenic with 21q11.2-q21.3. Here we report that the trisomy and the monosomy of this region alter locomotion, muscle strength, mass, and energetic balance. The expression profiling of skeletal muscles revealed global changes in the regulation of genes implicated in energetic metabolism, mitochondrial activity, and biogenesis. These genes are downregulated in Ts3Yah mice and upregulated in Ms3Yah mice. The shift in skeletal muscle metabolism correlates with a change in mitochondrial proliferation without an alteration in the respiratory function. However, the reactive oxygen species (ROS) production from mitochondrial complex I decreased in Ms3Yah mice, while the membrane permeability of Ts3Yah mitochondria slightly increased. Thus, we demonstrated how the Hspa13-App interval controls metabolic and mitochondrial phenotypes in muscles certainly as a consequence of change in dose of Gabpa, Nrip1, and Atp5j. Our results indicate that the copy number variation in the Hspa13-App region has a peripheral impact on locomotor activity by altering muscle function.     

Apoptosis in Down’s syndrome: lessons from studies of human and mouse models

Down syndrome (DS) is the most common chromosomal abnormality in humans. DS is characterized by a number of phenotypes, including the development of Alzheimer’s disease-like pathology and immunological, hematological and cardiovascular alterations. Apoptosis or programmed cell death is physiologically involved in development and aging, as well as in numerous pathological processes. Altered apoptosis has been proposed as a putative mechanism underlying many DS phenotypes. Evidence from human and animal studies indicates that apoptosis does not have a prominent role in the disturbances found in brain development in trisomy 21. However, alterations in apoptosis have been associated with neurodegeneration in the aging DS brain, with impairments in general growth and with immunological, cardiovascular and oncological alterations. Altered apoptosis in DS is likely to be the result of the interplay between several chromosome 21 (Hsa21) and non-Hsa21 genes. The interplay between these genes may affect physiological programmed cell death either directly, by modifying the activity of the apoptotic pathways, or indirectly, by inducing degeneration and rendering the cell more vulnerable to apoptosis-inducing factors.     

Down syndrome mouse models are looking up

A new mouse model of Down syndrome (DS) carries a copy of human chromosome 21 (Hsa21), in addition to a full complement of mouse chromosomes. In terms of the number of trisomic genes represented, this model, known as 'Tc1', is closer to the genetic background of DS than any previous model. The Tc1 model not only recapitulates several of the DS features present in other mouse models but also exhibits heart defects that are similar to those that make trisomy 21 the leading cause of congenital heart disease in humans. Many cells in adult Tc1 mice show mosaicism - that is, the Hsa21 is lost from some cells during development - increasing the complexity of analyses using this model. Tc1 mice provide a powerful tool for investigation of the pathogenesis of trisomy 21, and a platform for analysis of similarities and differences in the evolution of gene regulation.     

Increased Skin Tumor Incidence and Keratinocyte Hyper-Proliferation in a Mouse Model of Down Syndrome

Down syndrome (DS) is a genetic disorder caused by the presence of an extra copy of human chromosome 21 (Hsa21). People with DS display multiple clinical traits as a result of the dosage imbalance of several hundred genes. While many outcomes of trisomy are deleterious, epidemiological studies have shown a significant risk reduction for most solid tumors in DS. Reduced tumor incidence has also been demonstrated in functional studies using trisomic DS mouse models. Therefore, it was interesting to find that Ts1Rhr trisomic mice developed more papillomas than did their euploid littermates in a DMBA-TPA chemical carcinogenesis paradigm. Papillomas in Ts1Rhr mice also proliferated faster. The increased proliferation was likely caused by a stronger response of trisomy to TPA induction. Treatment with TPA caused hyperkeratosis to a greater degree in Ts1Rhr mice than in euploid, reminiscent of hyperkeratosis seen in people with DS. Cultured trisomic keratinocytes also showed increased TPA-induced proliferation compared to euploid controls. These outcomes suggest that altered gene expression in trisomy could elevate a proliferation signalling pathway. Gene expression analysis of cultured keratinocytes revealed upregulation of several trisomic and disomic genes may contribute to this hyperproliferation. The contributions of these genes to hyper-proliferation were further validated in a siRNA knockdown experiment. The unexpected findings reported here add a new aspect to our understanding of tumorigenesis with clinical implications for DS and demonstrates the complexity of the tumor repression phenotype in this frequent condition.     

Genetic analysis of Down syndrome facilitated by mouse chromosome engineering

Human trisomy 21 is the most frequent live-born human aneuploidy and causes a constellation of disease phenotypes classified as Down syndrome, which include heart defects, myeloproliferative disorder, cognitive disabilities and Alzheimer-type neurodegeneration. Because these phenotypes are associated with an extra copy of a human chromosome, the genetic analysis of Down syndrome has been a major challenge. To complement human genetic approaches, mouse models have been generated and analyzed based on evolutionary conservation between the human and mouse genomes. These efforts have been greatly facilitated by Cre/loxP-mediated mouse chromosome engineering, which may result in the establishment of minimal critical genomic regions and eventually new dosage-sensitive genes associated with Down syndrome phenotypes. The success in genetic analysis of Down syndrome will further enhance our understanding of this disorder and lead to better strategies in developing effective therapeutic interventions.     

Dosage of the Abcg1-U2af1 Region Modifies Locomotor and Cognitive Deficits Observed in the Tc1 Mouse Model of Down Syndrome

Down syndrome (DS) results from one extra copy of human chromosome 21 and leads to several alterations including intellectual disabilities and locomotor defects. The transchromosomic Tc1 mouse model carrying an extra freely-segregating copy of human chromosome 21 was developed to better characterize the relation between genotype and phenotype in DS. The Tc1 mouse exhibits several locomotor and cognitive deficits related to DS. In this report we analyzed the contribution of the genetic dosage of 13 conserved mouse genes located between Abcg1 and U2af1, in the telomeric part of Hsa21. We used the Ms2Yah model carrying a deletion of the corresponding interval in the mouse genome to rescue gene dosage in the Tc1/Ms2Yah compound mice to determine how the different behavioral phenotypes are affected. We detected subtle changes with the Tc1/Ms2Yah mice performing better than the Tc1 individuals in the reversal paradigm of the Morris water maze. We also found that Tc1/Ms2Yah compound mutants performed better in the rotarod than the Tc1 mice. This data support the impact of genes from the Abcg1-U2af1 region as modifiers of Tc1-dependent memory and locomotor phenotypes. Our results emphasize the complex interactions between triplicated genes inducing DS features.     

Postnatal lethality and cardiac anomalies in the Ts65Dn Down Syndrome mouse model

The Ts65Dn mouse is a well-studied model for Down syndrome (DS). The presence of the translocation chromosome T17¹⁶ (referred to as T65Dn) produces a trisomic dosage imbalance for over 100 genes on the distal region of mouse Chromosome 16. This dosage imbalance, with more than half of the orthologs of human Chromosome 21 (Hsa21), causes several phenotypes in the trisomic mice that are reminiscent of DS. Careful examination of neonates in a newly established Ts65Dn colony indicated high rates of postnatal lethality. Although the transmission rate for the T65Dn chromosome has been previously reported as 20%–40%, genotyping of all progeny indicates transmission at birth is near the 50% expected with Mendelian transmission and survival. Remarkably, in litters with maternal care that allowed survival of some pups, postnatal lethality occurred primarily in pups that inherited the T65Dn marker chromosome. This selective loss within 48 h of birth reduced the transmission of the marker chromosome from 49% at birth to 34% at weaning. Gross morphologic examination revealed cardiovascular anomalies, i.e., right aortic arch accompanied by septal defects, in 8.3% of the trisomic newborn cadavers examined. This is an intriguing finding because the orthologs of the DiGeorge region of HSA22, which are posited to contribute to the aortic arch abnormalities seen in trisomy 16 mice, are not triplicated in Ts65Dn mice. These new observations suggest that the Ts65Dn mouse models DS not only in its previously described phenotypes but also with elevated postnatal lethality and congenital heart malformations that may contribute to mortality.     

Segmental trisomy of mouse chromosome 17: introducing an alternative model of Down's syndrome

All of the mouse models of human trisomy 21 syndrome that have been studied so far are based on segmental trisomies, encompassing, to a varying extent, distal chromosome 16. Their comparison with one or more unrelated and non-overlapping segmental trisomies may help to distinguish the effects of specific triplicated genes from the phenotypes caused by less specific developmental instability mechanisms. In this paper, the Ts43H segmental trisomy of mouse chromosome 17 is presented as such an alternative model. The trisomy stretches over 32.5 Mb of proximal chromosome 17 and includes 486 genes. The triplicated interval carries seven blocks of synteny with five human chromosomes. The block syntenic to human chromosome 21 contains 20 genes.     

Molecular genetic analysis of Down syndrome

Down syndrome (DS) is caused by trisomy of all or part of human chromosome 21 (HSA21) and is the most common genetic cause of significant intellectual disability. In addition to intellectual disability, many other health problems, such as congenital heart disease, Alzheimer’s disease, leukemia, hypotonia, motor disorders, and various physical anomalies occur at an elevated frequency in people with DS. On the other hand, people with DS seem to be at a decreased risk of certain cancers and perhaps of atherosclerosis. There is wide variability in the phenotypes associated with DS. Although ultimately the phenotypes of DS must be due to trisomy of HSA21, the genetic mechanisms by which the phenotypes arise are not understood. The recent recognition that there are many genetically active elements that do not encode proteins makes the situation more complex. Additional complexity may exist due to possible epigenetic changes that may act differently in DS. Numerous mouse models with features reminiscent of those seen in individuals with DS have been produced and studied in some depth, and these have added considerable insight into possible genetic mechanisms behind some of the phenotypes. These mouse models allow experimental approaches, including attempts at therapy, that are not possible in humans. Progress in understanding the genetic mechanisms by which trisomy of HSA21 leads to DS is the subject of this review.     

An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model

Background

Down syndrome (DS), caused by trisomy of human chromosome 21 (HSA21), is the most common genetic cause of mental retardation in humans. Among complex phenotypes, it displays a number of neural pathologies including smaller brain size, reduced numbers of neurons, reduced dendritic spine density and plasticity, and early Alzheimer-like neurodegeneration. Mouse models for DS show behavioural and cognitive defects, synaptic plasticity defects, and reduced hippocampal and cerebellar neuron numbers. Early postnatal development of both human and mouse-model DS shows the reduced capability of neuronal precursor cells to generate neurons. The exact molecular cause of this reduction, and the role played by increased dosage of individual HSA21 genes, remain unknown.

Results

We have subcutaneously injected mouse pluripotent ES cells containing a single freely segregating supernumerary human chromosome 21 (HSA21) into syngeneic mice, to generate transchromosomic teratomas. Transchromosomic cells and parental control cells were injected into opposite flanks of thirty mice in three independent experiments. Tumours were grown for 30 days, a time-span equivalent to combined intra-uterine, and early post-natal mouse development. When paired teratomas from the same animals were compared, transchromosomic tumours showed a three-fold lower percentage of neuroectodermal tissue, as well as significantly reduced mRNA levels for neuron specific (Tubb3) and glia specific (Gfap) genes, relative to euploid controls. Two thirds of transchromosomic tumours also showed a lack of PCR amplification with multiple primers specific for HSA21, which were present in the ES cells at the point of injection, thus restricting a commonly retained trisomy to less than a third of HSA21 genes.

Conclusion

We demonstrate that a supernumerary chromosome 21 causes Inhibition of Neuroectodermal DIfferentiation (INDI) of pluripotent ES cells. The data suggest that trisomy of less than a third of HSA21 genes, in two chromosomal regions, might be sufficient to cause this effect.     

Mouse autosomal trisomy: two's company, three's a crowd.

Autosomal trisomy causes a large proportion of all human pregnancy loss and so is a significant source of lethality in the human population. The autosomal trisomy syndromes each have a different phenotype and are probably caused by the effects of specific genes that are present in three copies, rather than the normal two. Identifying these genes will require the application of classical genetic and new genome-manipulation approaches. Recent advances in chromosome engineering are now allowing us to create precisely defined autosomal trisomies in the mouse, and so provide new routes to identifying the critical, dosage-sensitive genes that are responsible for these highly deleterious, yet very common, syndromes.     

A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21

Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine β-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.     

Use of comparative physical and sequence mapping to annotate mouse chromosome 16 and human chromosome 21

Distal mouse chromosome 16 (MMU16) shares conserved linkage with human chromosome 21 (HSA21), trisomy for which causes Down syndrome (DS). A 4.5-Mb physical map extending from Cbr1 to Tmprss2 on MMU16 provides a minimal tiling path of P1 artificial chromosomes (PACs) for comparative mapping and genomic sequencing. Thirty-four expressed sequences were positioned on the mouse map, including 19 that were not physically mapped previously. This region of the mouse:human comparative map shows a high degree of evolutionary conservation of gene order and content, which differs only by insertion of one gene (in mouse) and a small inversion involving two adjacent genes. "Low-pass" (2.2x) mouse sequence from a portion of the contig was ordered and oriented along 510 kb of finished HSA21 sequence. In combination with 68 kb of unique PAC end sequence, the comparison provided confirmation of genes predicted by comparative mapping, indicated gene predictions that are likely to be incorrect, and identified three candidate genes in mouse and human that were not observed in the initial HSA21 sequence annotation. This comparative map and sequence derived from it are powerful tools for identifying genes and regulatory regions, information that will in turn provide insights into the genetic mechanisms by which trisomy 21 results in DS.