Mouse autosomal trisomy
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. neuploidy is a condition in which there are more or fewer chromosomes than an exact multiple of the haploid number for that species. In diploid species, such as mouse and human, if an individual chromosome is present in three, rather than the normal two, copies, it is ‘trisomic’, or if present in one copy it is ‘monosomic’. Wholechromosome aberrations generally occur because of meiotic non-disjunction. Duplications, deletions and other rearrangements of parts of chromosomes can also produce an unbalanced genetic complement, such as three copies or one copy of a particular region; in these cases, the individual can have an aneuploid or a euploid number of chromosomes. In mouse and human, the sex chromosome aneuploidies are less severe than the autosomal aneuploidies, providing the individual has at least one X chromosome. No autosomal monosomies are viable, presumably because at least two ‘doses’ of a subset of the genes on each autosome are required for normal development. The human autosomal trisomies are relatively common – it is estimated that 20% of human conceptuses are trisomic – but all have a very high degree of morbidity and mortality and most abort early in embryogenesis1. Only three human autosomal trisomies routinely survive beyond birth: trisomy 13, 18 and 21, and only trisomy 21 individuals survive beyond early childhood.
Dissecting the molecular genetics of trisomy syndromes The paradigm for the study of human trisomy syndromes, and attempts to find the causal genes, is Down syndrome, which arises from trisomy 21 and occurs approximately once in 600 newborns2. The syndrome is a complex disorder with a highly variable phenotype and is the most common known cause of mental retardation. Most aspects of 0168-9525/99/$ – see front matter © 1999 Elsevier Science All rights reserved. PII: S0168-9525(99)01743-6
the syndrome are probably caused by having an extra copy of a set of normal but ‘dosage-sensitive’ genes on chromosome 21 (Hsa21)* that give an aberrant phenotype when present in three copies. Standard genetic routes of investigation, such as linkage analysis, will not help to identify these causal genes; thus, innovative approaches are needed to deconstruct the molecular genetics of aneuploidy syndromes. The mouse autosomal trisomies are valuable aids for identifying dosage-sensitive genes, and for understanding the relationship between gene dosage and phenotype3. By using mouse trisomies that are well defined at the molecular and phenotypic levels, and new chromosome engineering technologies, we can assay chromosome regions for dosage-sensitive effects and dissect regions of interest to map aneuploidy syndrome loci. We can then move to more-conventional transgenic approaches to identify candidate genes4. Mice are not mini-humans and genes that are dosage sensitive in one organism might have no effect in the other. However, mouse trisomy analysis, in combination with human studies, will provide a powerful system for understanding how and why the aneuploidy syndromes arise in both species.
Primary, whole-chromosome, trisomy The normal diploid chromosomal complement of the laboratory mouse is 40 [19 pairs of acrocentric (singlearmed) autosomes and one pair of sex chromosomes]. The condition of having a whole, extra, freely segregating chromosome has been referred to as ‘primary’ trisomy, whereas a secondary trisomy refers to the situation in *We are using the notation ‘Hsa’ to denote a human chromosome (from Homo sapiens) and Mmu to denote a mouse chromosome (Mus musculus).
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Diana Hernandez [email protected]
Elizabeth M.C. Fisher [email protected]
Department of Neurogenetics, Imperial College School of Medicine (St Mary’s), Norfolk Place, London, UK W2 1PG. 241
Mouse autosomal trisomy
which two identical chromosome arms are joined to a common centromere, creating an isochromosome. Because trisomies are seen only rarely in the mouse, breeding schemes have been developed to produce trisomic progeny at a reasonably high frequency. If two non-homologous autosomes fuse at their centromeres, they produce a biarmed metacentric chromosome, known as a Robertsonian (Rb) chromosome or Robertsonian translocation, and these chromosomes occur naturally in some mouse populations. A mouse with one Rb metacentric chromosome has 39 chromosomes, but a complete complement of 40 chromosome arms and a complete diploid gene set; thus, the mouse is phenotypically normal. Mating schemes using Rb mice have been devised to produce progeny that are normal, trisomic or monosomic for the Rb chromosome constituents (Fig. 1)5,6. All mouse autosomal trisomies are retarded in growth and development. Most are lethal before 16 days gestation, very few survive to birth and only trisomy 19 mice have consistently been seen to survive beyond birth (see Table 1 for summary). Phenotypic variability can be attributed to maternal and embryonic strain effects7,8. Trisomy 16 (Ts16) is the most studied of the mouse autosomal trisomies because human chromosome 21 is largely syntenic with a region of mouse chromosome 16; thus, until recently, whole trisomy 16 was the best mouse model of Down syndrome. However, this model has two major disadvantages: the gene content of Hsa21 and Mmu16 are similar but far from identical; also, trisomy 16 mice die at birth, which precludes them from studies of adult physiology, behaviour and so on (Table 2).
of partial trisomy in which only a portion of a chromosome is trisomic; in this case individuals are euploid (Fig. 2). Mice with these and more complex chromosome rearrangements can be generated by radiation and chemical mutagenesis, and many different examples are known and well documented9,10. Partial trisomies can be less severe than the whole-chromosome trisomies, presumably depending on the gene content of the triplicated region; nevertheless, most partially trisomic mice die during embryogenesis. Making phenotype–genotype correlations can often be complicated in the partial trisomies because of partial monosomy for a second chromosomal region, or abnormal gene products being produced from translocation breakpoints. Two partial trisomies that have generated a lot of recent interest as potential models for aspects of Down syndrome are the Ts65Dn and the Ts1Cje mouse11–13. Ts65Dn mice are aneuploid (41 chromosomes) because they possess an extra small marker chromosome composed of the centromere of chromosome 17 (Mmu17) and a large portion of Mmu16 that has homology with Hsa21 (Refs 11, 12). Ts1Cje mice carry a chromosome rearrangement in which a smaller region of Mmu16 has translocated onto Mmu12; thus, the mice are euploid but partially trisomic for Mmu16 (Ref. 13). Ts65Dn and Ts1Cje mice survive into adulthood and they have different behavioural and other phenotypic abnormalities. Phenotype–genotype comparisons between these animals might be helpful for mapping dosage-sensitive genes on Mmu16 (Refs 11–14 and Table 2).
Engineering new mouse trisomies Partial, tertiary, segmental trisomy ‘Partial’ trisomy refers to any situation in which only part of a chromosome is trisomic; partial trisomy individuals can be diploid or aneuploid, depending on the chromosome rearrangement. One type of partial trisomy is ‘tertiary’ trisomy, which arises when a product of a reciprocal translocation chromosome is inherited, giving genetically unbalanced progeny. ‘Segmental’ trisomy is another type
FIGURE 1. Producing mice with whole chromosome trisomy (and monosomy) Mouse heterozygous for a Robertsonian translocation
Mouse with 40 acrocentric chromosomes
Existing mouse trisomies have a long research history, and they can now be supplemented with mice created by new chromosome-engineering technologies that will allow us to choose exactly which chromosomal region is trisomic, whether the DNA is of mouse or other origin and whether the trisomy is due to segmental trisomy or the presence of an extra, freely segregating chromosome. Thus, we can make precise correlations of genotype and phenotype on defined genetic backgrounds for the identification and, ultimately, isolation of dosage-sensitive genes. Three approaches are currently in use: making new use of an old technology, microcell-mediated chromosome transfer (so far used for placing extra, freely segregating human chromosome sequences into mice); creating mammalian artificial chromosomes; and using the Cre–loxP system to generate chromosome rearrangements (so far used for creating segmental trisomy and monosomy in mice). All three approaches depend on engineering the trisomy in a mouse host environment, usually mouse embryonic stem (ES) cells, and then using conventional transgenic techniques to create strains of trisomic mice.
Microcell-mediated chromosome transfer Disomy Trisomy
A wild-type mouse is mated to a mouse that is heterozygous for a Robertsonian translocation. Because the chromosomes involved in the Rb translocation are joined at the centromeres, in a proportion of cases non-disjunction produces unbalanced gametes. Other schemes involve mating mice that are doubly heterozygous for two different Rb chromosomes6. Two different chromosome pairs are shown in blue and red.
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Microcell-mediated chromosome transfer (MMCT) is a technology that has been in use since the 1970s for transferring donor chromosomes into recipient cells to create cell hybrids15. Donor cells containing the chromosome to be transferred are arrested in metaphase and micronucleated, creating microcells that consist of intact chromosomes surrounded by nuclear and cell membranes. The microcells are fused to recipient cells, and a proportion of the donor chromosomes becomes stably incorporated
Mouse autosomal trisomy
TABLE 1. Phenotype of the mouse autosomal primary trisomies Chromosome 1
2 3 4 5
6 7 8
9 10 11 12
17 18 19
From slight developmental retardation to hypoplasia and holoprosencephaly combined with aprosopia or cyclopia. Some authors have found no specific abnormalities, while others found fetuses with severe facial dysplasia and microgenia, brain and eye defects, especially in the lens and optic cup. Embryos are severely developmentally retarded and hypoplastic, failure of neural plate closure is common. Very small embryos severely growth retarded and disorganized. Moderate developmental retardation with no obvious abnormalities. Major developmental block at around the time of organogenesis. Embryos are small and severely hypoplastic, they fail to rotate, their neural tube remains open and there are no signs of eye or otic placodes. No heartbeat can be detected and there is very poor vascularization of the yolk sac. Growth retardation ranging from moderate to severe, eye defects that range from monolateral coloboma to bilateral anophthalmia, also cleft palate. Growth and developmental retardation. There is general growth retardation, with specific defects such as reduced cranial region and non-closure of the neural tube in some embryos and dilated heart chambers in others. The developmental block may be due to the failure of chorio-allantoic placental function. General retardation, cranioschisis, expanded heart primordium, failure of allantoic growth and neural tube closure with exencephaly, head hypoplasia. Slight developmental delay, very moderate hypoplasia and no gross external malformations. Some show heart defects. Severe retardation and hypoplasia. In severe cases, all that remains of the embryos is a disorganized degenerative mass. Little developmental retardation; embryos can survive up to or near to term in certain mouse strains. They have a very specific defect: marked exencephaly and microphthalmia. The defect of the cranial vault is always complete and the brain protrudes beyond the cranium. Cerebral lobes are well developed. Secondary effects due to the failure of neural tube closure include eye and skull defects. Congenital cardiovascular defects have been found. Death of the embryos is most likely caused by the hypoplasia and differentiation arrest of the circulatory system of the embryonic part of the chorio-allantoic placenta. Developmentally delayed, marked transient oedema of the neck and back, delayed ossification and cleft palate. Specific cardiovascular malformations. Size reduction in the spinal cord due to a reduction of the neuropil in the grey matter, atrophy of the dendritic arborization in the neuropil and hence a severe restriction of interneuronal connectivity. General retardation and moderate hypoplasia, some show exencephaly but this is less severe than that of Ts12 embryos, as the neural tube is closed up to the hindbrain/midbrain junction and only the forebrain remains open. Cardiovascular defects of the ventricular septal defect type occur. Histologically, retardation of differentiation of the thymus and gonads is often seen, with necrosis of the ovaries being the most prominent. Severe retardation. Some embryos show specific malformations of the neural tube with abnormal curve in the breast or lumbar regions and are generally severely retarded. Ts15 is the most common chromosomal aberration in spontaneous and induced leukaemias in mice. Small in size, have short necks, shortened snouts, small heads and open eyelids (at birth). Internally the most obvious defects are of cardiac origin, the most common being atrio-ventricular septal defects. Embryos show retarded brain development and small thymuses. An extensive literature exists on the trisomy 16 mice (see Table 2 and references for further details). Disorganization and runting prior to death in certain genetic backgrounds. In backgrounds with longer survival times (12–13 dpc) there are specific caudal malformations with local haemorrhages and necrosis of the tail. From early lethality to birth with no other abnormality other than cleft palate, depending on genetic background. The most obvious feature of these mice is developmental and growth delay with some showing cleft palate and specific atrophy of the ovaries. Altered development of the visual cortex and behavioural abnormalities have been reported in rare surviving Ts19 mice.
Until 15 dpc
Until 10 dpc 10–13 dpc 12–13 dpc Until 11 dpc
34 5, 34 5, 34 34
Until 10 dpc Until 10 dpc
Until 12 dpc
15–20 dpc Until 11 dpc Up to birth
34, 35 6, 34 34, 35
34, 35, 38
34, 35, 39
Until 10 dpc
Few hours post-natally
34, 35, 45
12 dpc–birth Up to 3 weeks post-natally
34, 35, 45 34, 46
Abbreviation: dpc, days post conception.
and segregates freely within the recipient nuclei. Selection can then be applied for a particular chromosome of interest. Once an MMCT cell line has become established, the extra chromosome is often maintained without the need for continuous culturing in selection. The MMCT protocol has been modified by irradiating the microcells before fusion to the recipient cell line, in order to fragment the donor chromosomes (‘irradiation MMCT’, ‘XMMCT’, see Refs 16, 17 and references therein; Fig. 3). Tomizuka and colleagues presented the first report of using MMCT in combination with ES-cell transgenic technologies, to introduce freely segregating stable human chromosome fragments (Hsa2, Hsa14) or a complete chromosome (Hsa22), into chimeric mice18. The ES-cellderived components of these mice have the normal diploid number of 40 mouse chromosomes plus an extra chromosome that is human. Thus, they have three copies of the genes that are present on the human chromosomes (i.e. two mouse homologues and one human homologue). In this study18, these ‘transchromosomal’ chimeric mice expressed genes from the human chromosomes and the Hsa2 fragments transmitted through the germline of these mice.
The MMCT approach does not allow the boundaries of the chromosome fragments to be determined precisely because the chromosomes break randomly (and, in the case of XMMCT, with a frequency that is largely dependent on irradiation dose). However, transchromosomal ES cell lines can be analysed in advance of making chimeric mice, to select those cell lines that contain trisomic regions of interest. In our own hands, the XMMCT protocol works well for placing freely segregating Hsa21 minichromosomes into chimeric mice17. Chromosomes can be engineered for specific truncations at desired locations by targeting telomere sequences at internal chromosome sites, using homologous recombination. Relatively high frequencies of homologous recombination can be achieved in mouse ES cells; targeting human chromosomes has been achieved in human cells and human–mouse-cell hybrids, but so far it has proven inefficient because of the low frequency of homologous recombination in these cells17,19. (Possibly, human ES cells could provide an efficient environment for targeting human sequences.) To overcome the current low efficiency in targeting human sequences, an alternative cell system can be used: the chicken pre-B cell line, DT40. TIG June 1999, volume 15, No. 6
Mouse autosomal trisomy
TABLE 2. Phenotype of three mouse trisomies involving chromosome 16a Ts16 mouse
Chromosomal anomaly Trisomy of the whole of Mmu16 (aneuploid)
Trisomy of Mmu16 from App to Mx2 (aneuploid)
Trisomy of Mmu16 from Sod1 to Mx1 (euploid)
Small in size; mild hydrocephalus. Early onset obesity
No gross differences in appearance compared to controls
Hyperactivity, and early onset tremors
Normal activity, even hypoactivity
Gross appearance Small in size; defects in skeletal morphology; short neck, small head, foreshortened snout; open eyelids
Activity Nervous system development Retarded brain development; reduced cortical surface, decreased and delayed proliferation of neurons; altered electrophysiology of some neurons
Developmental delay; mice develop sensorimotor milestones later than controls
Brain pathology Decreased catecholaminergic system markers, except for increased DOPA decarboxylase and catechol-O-methyl transferase; decreased (?) cholinergic system markers; decreased serotonergic system markers; decreased numbers of basal forebrain cholinergic neurons (BFCNs) generated
Selective age-dependent loss and cellular atrophy of BFCNs (though starting numbers are normal); deficiencies in the synaptic transmission of the central b-noradrenergic system, selective for specific brain areas (cortex and hippocampus); hypertrophy of astrocytes; unaffected cholinergic, serotonergic and catecholaminergic neuronal morphology or numbers
No loss or degeneration of BFCNs, no neuronal atrophy
Cognitive impairment, especially of spatial learning memory both short- and long-term; deficient in both spatial working and reference memory; altered long-term potentiation
Impairment of spatial learning, deficits, especially in learning flexibility
Cardiovascular development Congenital heart defects; myocardial defect which influences shape of the heart tube producing atrio-ventricular septal defect
Immune system Small thymus; reduced stem, and other, cell numbers; abnormal cell responses, e.g. delayed maturation of lymphocytes
Other systems Liver abnormalities
Possible anomalies in digestive function and amino acid metabolism
Fertility Male infertility, females subfertile
Both males and females fertile
Lives to adulthood
Lives to adulthood
Life expectancy Death at birth a
Taken from Refs 11–13, 41–44, 47–60.
In this cell line, levels of homologous recombination into human chromosomes have been reported to be 1000- to 10 000-fold higher than those seen in mouse erythroid leukemia (MEL)–human hybrid cells, and are similar to those of mouse ES cells20,21. To achieve DNA targeting in the DT40 system, human chromosomes are transferred from donor cells into the chicken cells using MMCT. This creates chicken–human hybrid lines that can be transfected with conventional targeting constructs to obtain the desired changes to the human chromosomes, at high rates. The engineered (for example, truncated) chromosomes can then be reintroduced into mammalian cells, again using MMCT, because DT40 cells are good recipients as well as donors in the MMCT protocol20. This system has been used successfully, both to introduce targeted mutations and to create truncations in human chromosomes and mammalian artificial chromosomes20,21. Kuroiwa et al.22 have used the DT40 cell line to truncate Hsa22: they transferred Hsa22 from a mouse–human hybrid into DT40 cells and engineered a 244
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truncation of this chromosome by targeting telomere sequences into the Lif locus22. DT40 cells are then a useful system for the precise engineering of human chromosomes in vitro, prior to the reintroduction of the mutated chromosome into mammalian cells, either for in vitro studies or for the production of mouse chimeras.
Mammalian artificial chromosomes The prime motivations for developing mammalian artificial chromosomes (MACs) are the need to determine the chromosomal components, such as centromeres and origins of replication, that are essential for chromosome function (including correct disjunction), and the need to find new delivery vectors for gene therapy. Such vectors would allow large inserts to be transferred into host cells, without chromosomal position effects on gene transcription or insertional mutagenesis of the host genome. MACs are being created using a ‘top-down’ approach, in which a normal chromosome is deleted to the minimum functioning unit, or a ‘bottom-up’ approach, in which different chromosomal components are ligated together (reviewed
Mouse autosomal trisomy
in Ref. 21). MACs present a route for creating partially trisomic mice that contain defined regions of either mouse or human DNA. To be useful, MACs need to be engineered precisely and efficiently, and they need a routinely successful protocol for transfecting mouse ES cells to create transchromosomal trisomic mice. One approach to working with MACs is to use the DT40 chicken lymphoid cell line in combination with the MMCT protocol, as described above. MACs can be introduced into DT40 cells and, in this environment, they can be targeted efficiently at specific sites by homologous recombination23. Transferring MACs into DT40 or ES cells requires a protocol that will not shear the DNA and that will enable the stable incorporation of the extra chromosome into the nucleus; thus, MMCT has become a method of choice20. Human minichromosomes of 4 Mb and 15 Mb, based on deleted Y chromosomes, have been introduced into mouse ES cells by MMCT (Ref. 24). In both cases the minichromosomes rearranged and were lost in the absence of selection. However, one 4 Mb mini-chromosome, which rearranged to incorporate mouse centromeric sequences, was maintained stably without selection, and is potentially a new vector for creating trisomic mice with human or mouse DNA regions of interest24.
Chromosome engineering with the Cre–loxP system Cre is a bacteriophage DNA recombinase that recognizes a 34 bp sequence, called the loxP site, and efficiently catalyzes reciprocal conservative DNA recombination between two loxP sites (reviewed in Ref. 25). loxP sites consist of two 13 bp inverted repeats, flanking an 8 bp non-palindromic core region that gives the sequence directionality. Recombination between two directly repeated loxP sites results in the excision of the intervening DNA as a covalently closed circle (Fig. 4a), while recombination between loxP sites in inverted orientations results in the inversion of the intervening DNA (Fig. 4b). Thus, loxP sites can be used to produce a deletion when placed directly repeated in cis along a chromosome, whereas if the sites are indirectly repeated, the DNA between the sites is inverted. Cre not only catalyzes intramolecular (in cis), but also intermolecular (in trans) DNA recombination; therefore, it can be used to produce chromosomal rearrangements between homologous and non-homologous chromosomes. Because Cre recombination is reciprocal, placing loxP sites in the same orientation at distinct loci on chromosome homologues (i.e. in trans) results in a deletion on one chromosome and a duplication of the targeted interval on the other25,26 (Fig. 4c). Therefore, this can generate defined segmental aneuploidies of the targeted chromosome. Alternatively, placing loxP sites in the same orientation at distinct loci on non-homologous chromosomes will result in translocation between these chromosomes, at the loxP sites (Fig. 4d). The first step in engineering chromosomal rearrangements in mouse is to target loxP sites by homologous recombination at the desired endpoints of the rearrangement, in ES cells, and then transiently express Cre in these cells in order to catalyze recombination between the two sites in vitro25,26. Using the Cre–loxP system, with loxP sites placed in cis on Mmu11, mice with a 1 Mb duplication on Mmu11 were created. These have a well-defined phenotype of corneal hyperplasia and thymic tumours, presumably because of
FIGURE 2. Different types of partial trisomy (a)
b c d
b c d
Partial trisomy Mouse is aneuploid
b c d
b c b c d
Segmental trisomy Mouse is euploid
Partial monosomy Tertiary trisomy
Tertiary Balanced trisomy carrier Partial monosomy
The mouse in (a) has 41 chromosomes and is partially trisomic for a portion of one chromosome. The mice in (b) with reciprocal translocations that are balanced carriers and phenotypically normal have 40 chromosomes and can be bred to produce offspring with tertiary trisomy. In (c), mice with segmental trisomy have 40 chromosomes and are trisomic for a portion of one chromosome.
the presence of three copies of a dosage-sensitive gene(s) within the duplication26,27. Also, using two loxP sites placed in trans on different non-homologous chromosomes, two groups have engineered new translocations with precise breakpoints between Mmu12 and 15, and between Mmu2 and Mmu13, in ES cells28,29. When two loxP sites are on the same chromosome homologue, that is, in cis, the frequencies of recombination between them on different chromosomes are higher than recombination in trans. The frequency of Cre–loxP mediated chromosome rearrangements in trans between two homologues can be increased by a new technique: targeted meiotic recombination (TAMERE)30, which takes
FIGURE 3. Microcell-mediated chromosome transfer
Arrest in metaphase with colcemid Donor cell Photograph of Centrifuge micronucleated cells in the presence of cytochalasin B Collect microcells Irradiate if XMMCT
Fuse to recipient cells Apply selection if appropriate
Chimeric mouse Inject into blastocysts (if recipient is ES cell)
Collect hybrid clones
Irradiation microcell-mediated chromosome transfer (XMMCT) is a variation of microcell-mediated chromosome transfer (MMCT), in which microcells are irradiated prior to fusion with the recipient cell.
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Mouse autosomal trisomy
Figure 4. Cre-mediated recombination between lox P sites With lox P sites placed in cis (a) lox P sites directly repeated C
D lox P
B lox P
(b) lox P sites invertedly repeated
Inversion lox P
With lox P sites in trans (c) lox P sites targeted to chromosome homologues A
lox P A
(d) lox P sites targeted to non-homologous chromosomes
lox P A
Z lox P
Express Cre A
lox P B
(a) When lox P sites are directly repeated in cis on one chromosome, recombination results in a deletion between the sites with a lox P sequence left in place. (b) When lox P sites are invertedly repeated in cis on one chromosome, recombination results in an inversion of the DNA between the sites, and both lox P sequences remain in the chromosome. (c) If lox P sites are directly repeated in trans, on two different chromosome homologues, then recombination produces a deletion on one chromosome and a duplication on the other, with maintenance of lox P sites as shown. The efficiency of this process is greatly increased by the TAMERE technique. (d) If lox P sites are directly repeated in trans, on two non-homologous chromosomes, then recombination produces two reciprocal translocation products, and the lox P sites remain as shown.
advantage of the enhanced recombination that results from chromosomes that are pairing during meiosis. Thus, Cre–loxP-mediated recombination in trans, between chromosome homologues, occurs at a higher frequency in vivo by expressing Cre under the control of a testis/zygotenespecific promoter. Two strains of mice, each carrying one loxP site in different sites in the Hoxd complex, were crossed30 and male progeny were selected that were transheterozygous for both alleles and hemizygous for the Cre transgene. Samples of testis genomic DNA from these males were shown to contain either a duplication or a deletion of the region between the two loxP sites. Mating these males to wild-type females produced progeny with either the duplicated allele or the deleted allele30. It is not known how efficient TAMERE will be over long distances. Thus, the Cre-loxP system allows us to create new mouse strains that carry precisely defined chromosome translocations and segmental trisomies. Experimental designs that make use of in vivo recombination mechanisms will significantly increase the efficiency with which such mice can be produced.
different Rb translocations of Mmu19 lived for 20 weeks and produced a pup that died shortly after birth31). There are very few reports of the germline transmission of an extra, freely segregating, partially trisomic mouse chromosome32, but a notable exception is the Ts65Dn mouse, which transmits the extra, freely segregating chromosome through the female germline11,12. Males are infertile, probably because spermatogenesis arrests in metaphase I owing to the presence of the extra chromosome. Another example of germline transmission of an extra chromosome in the mouse has been described from the MMCT studies of Tomizuka et al.18 Four transchromosomal male chimeras, derived from an XY ES cell line carrying a small portion of Hsa2, produced 316 pups, of which two carried Hsa2 fragments. Four transchromosomal female chimeras derived from a 39, X0 ES cell line produced 67 pups, of which 22 carried Hsa2 fragments. Thus, transmission of an extra, partially trisomic or human chromosome is possible through the mouse germline, although transmission appears to be more efficient through the female germline (as is similarly the case with human trisomy 21).
Germline transmission of trisomic chromosomes If a mouse is trisomic but euploid (e.g. a segmental trisomy) then, depending on the chromosomal rearrangement, germline transmission might present no difficulties. However, mice with primary trisomy die before reproducing (although one female trisomy 19 mouse carrying two 246
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Finding dosage-sensitive genes By precisely manipulating regions of trisomy, we can assay smaller and smaller genomic segments for the presence of dosage-sensitive genes, until it becomes feasible to move to more conventional transgenic approaches to pinpoint
Mouse autosomal trisomy
single genes and determine their effects when present in three doses. Liu et al.27 have shown that a duplication constructed by Cre-loxP recombination rescues the haplolethality of a deletion they created in Mmu11. Because the duplicated region is only 1 Mb, is it now feasible to use a yeast artificial chromosome (YAC) transgenic approach to find the causal dosage-sensitive gene(s). Smith et al.4,33 assayed a 2 Mb region of Hsa21 for such genes using overlapping YACs injected into the pronucleus of mouse oocytes. A subset of the transgenic mice exhibited learning and other neurological defects. Using a combination of different YAC transgenic mice, the authors concluded that the phenotype is most likely due to the mice having three functional copies (two mouse, one human) of the Hsa21 gene minibrain (Ref. 4). It would be possible to test this further by ablating one of the three minibrain genes in these mice and determining if the wild-type phenotype was rescued.
References 1 Shaffer, L.G. et al. (1998) Systematic search for uniparental disomy in early fetal losses: the results and a review of the literature. Am. J. Med. Genet. 79, 366–372 2 Hernandez, D. and Fisher, E.M.C. (1996) Down syndrome genetics: unravelling a multifactorial disorder. Hum. Mol. Genet. 5, 1411–1416 3 Gearhart, J.D. et al. (1987) Developmental consequences of autosomal aneuploidy in mammals. Dev. Genet. 8, 249–265 4 Smith, D. et al. (1997) Functional screening of 2Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome. Nat. Genet. 16, 28–36 5 Gropp, A. et al. (1974) Trisomy in the fetal backcross progeny of male and female metacentric heterozygotes of the mouse. I. Cytogenet. Cell Genet. 13, 511–535 6 Gropp, A. et al. (1975) Systematic approach to the study of trisomy in the mouse. II. Cytogenet. Cell Genet. 14, 42–62 7 Epstein, C. (1990) Genetic control of survival of murine trisomy 16 fetuses. Teratology 42, 571–580 8 Demczuk, S. and Vekemans, M. (1993) Genetic analysis of the maternal factors controlling the survival of trisomy 16 mouse fetuses. Teratology 47, 311–319 9 Stubbs, L. et al. (1997) Generation and characterization of heritable reciprocal translocations in mice. Methods 13, 397–408 10 Cattanach, B.M. et al. (1993) Large deletions and other gross forms of chromosome imbalance compatible with viability and fertility in the mouse. Nat. Genet. 3, 56–61 11 Davisson, M. et al. (1993) Segmental trisomy as a mouse model for Down syndrome. Prog. Clin. Biol. Res. 384, 117–133 12 Reeves, R. et al. (1995) A mouse model for Down syndrome exhibits learning and behaviour deficits. Nat. Genet. 11, 177–184 13 Sago, H. et al. (1998) Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc. Natl. Acad. Sci. U. S. A. 95, 6256–6261 14 Kola, I. and Hertzog, P. (1998) Down syndrome and mouse models. Curr. Opin. Genet. Dev. 8, 316–321 15 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. U. S. A. 74, 319–323 16 Koi, M. et al. (1993) Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11. Science 260, 361–364 17 Hernandez, D. et al. (1999) Transchromosomal mouse embryonic stem cell lines that contain a freely segregating whole or partial human chromosome 21. Hum. Mol. Genet. 8, 923–933 18 Tomizuka, K. et al. (1997) Functional expression and germline transmission of a human chromosome fragment in chimeric mice. Nat. Genet 16, 133–143 19 Itzhaki, J.E. et al. (1997) Construction by gene targeting in human cells of a ‘conditional’ CDC2 mutant that rereplicates its DNA. Nat. Genet. 15, 258–265 20 Dieken, E.S. et al. (1996) Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids. Nat. Genet. 12, 174–182
The future The research presented here spans traditional mouse genetics and the studies of trisomic embryos and runs through to present-day chromosome engineering projects. It is likely that chromosome engineering will become considerably more refined in the near future as, for example, the targeting efficiency of the DT40 system is combined with Cre–loxP manipulation and MMCT or TAMERE to create trisomies to order. We need these imaginative approaches in the mouse to begin to understand the autosomal aneuploidy syndromes – the most common, and possibly the most complex, of disorders.
Acknowledgements We thank M. Davisson and G. Truett for replies to our MGI Bulletin Board query, and P. Biggs and C. Huxley for critical reading of portions of the review. D.H. is funded by the Wellcome Trust.
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TIG June 1999, volume 15, No. 6