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Journal List > Genetics > v.180(1); Sep 2008

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Genetics. 2008 September; 180(1): 51–59.
doi: 10.1534/genetics.108.092312.
PMCID: PMC2535701
Copyright © 2008 by the Genetics Society of America
Inducing Segmental Aneuploid Mosaicism in the Mouse Through Targeted Asymmetric Sister Chromatid Event of Recombination
Arnaud Duchon,*† Vanessa Besson,*† Patricia Lopes Pereira,*† Laetitia Magnol,*† and Yann Hérault*†‡1
*Université d'Orléans, UMR6218, Molecular Immunology and Embryology, 45071 Orléans Cedex 2, France, †Centre National de la Recherche Scientifique, UMR6218, Immunologie and Molecular Embryology, 45071 Orléans Cedex 2, France and ‡Centre National de la Recherche Scientifique, UPS44, Transgenese, Archivage Animaux Modèles, Institut de Transgenose, 45071 Orléans Cedex 2, France
1Corresponding author: CNRS, Institut de Transgénose 3B rue de la Férollerie, 45071 Orléans Cedex 2 France. E-mail: herault/at/cnrs-orleans.fr
Communicating editor: N. Arnheim
Received June 6, 2008; Accepted July 10, 2008.
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Abstract
Loss or gain of whole chromosomes, or parts of chromosomes, is found in various pathological conditions, such as cancer and aneuploidy, and results from the missegregation of chromosomes during cellular division or abnormal mitotic recombination. We introduce a novel strategy for determining the consequences of segmental aneuploid mosaicism, called targeted asymmetric sister chromatin event of recombination (TASCER). We took advantage of the Cre/loxP system, used extensively in embryonic stem cells for generating deletions and duplications of regions of interest, to induce recombination during the G2 phase. Using two loxP sites in a Cis configuration, we generated in vivo cells harboring microdeletions and microduplications for regions of interest covering up to 2.2 Mb. Using this approach in the mouse provides insight into the consequences of segmental aneuploidy for homologous regions of the human chromosome 21 on cell survival. Furthermore, TASCER shows that Cre-induced recombination is more efficient after DNA replication in vivo and provides an opportunity to evaluate, through genetic mosaics, the outcome of copy number variation and segmental aneuploidy in the mouse.
 
VARIATIONS in copy number are an important source of modifications in the genome, contributing to the diversity of phenotypes and to disease (Shaw and Lupski 2004; Gonzalez et al. 2005; Aitman et al. 2006; Fellermann et al. 2006; Sebat et al. 2007; Sharp et al. 2007, 2008; Hollox et al. 2008). They correspond to segmental duplications or deletions and represent rare variants or polymorphic events in the population (Feuk et al. 2006a; Redon et al. 2006; Kidd et al. 2008). As a consequence, genetic expression could change either directly, for genes located inside the region, or indirectly through positional effects for genes located outside (Lupski and Stankiewicz 2005; Feuk et al. 2006a,b; Freeman et al. 2006).
Complete loss of a chromosome often induces more severe consequences with cellular dysfunction, such as tumorigenesis, impaired viability, such as spontaneous abortion, or aneuploid syndromes such as Down syndrome (DS) (Robinson et al. 1999, 2001; Hassold and Hunt 2001; Hassold et al. 2007). Nevertheless, aneuploid mosaicism has been described for almost all the chromosomes in humans and confined chromosomal mosaicism affects ~2% of viable pregnancies (Hahnemann and Vejerslev 1997; Kalousek 2000). It originates during cell division with the missegregation of a chromosome; while one cell will receive an additional chromosome leading to trisomy, the second daughter cell will be monosomic. Somehow, the distribution of mosaicism depends on the timing, the cell lineage, the viability of the aneuploid cells, and the chromosome or the region involved. Generalized chromosomal mosaicisms, induced early during embryogenesis, exist but are less frequent and often linked to pathological conditions. For example, in DS, mosaicism represents 1–2% of cases (Mikkelsen 1977), whereas the complete absence of the HSA21 is almost incompatible with life (Katz-Jaffe et al. 2004, 2005). Somatic aneuploid mosaicism has been described in blood cells (Cheung et al. 2007) and in normal brain with a frequency of 1.25–1.45% per chromosome and a percentage of aneuploid cells >30% (Rehen et al. 2001, 2005; Kingsbury et al. 2005, 2006; Yurov et al. 2008). Several hypotheses have been drawn to explain the presence and the outcome of such aneuploid cells (Kingsbury et al. 2006) but no clear evidence has been documented.
In vivo, the induction of segmental aneuploidy has been achieved by using the Cre/loxP recombinase system in several species (for review see Lewandoski and Martin 1997; Brault et al. 2006). In the mouse, a large panel of Cre transgenes are described to generate segmental deletion, duplication, inversion, and translocation (Kmita et al. 2002; Spitz et al. 2005; Wu et al. 2007). For such large fragments, specific promoters have been used to direct the expression of the recombinase during meiosis in the targeted meiotic recombination (TAMERE), (Herault et al. 1998; Kmita et al. 2002; Genoud et al. 2004; Zakany et al. 2004; Tarchini and Duboule 2006), in different tissues (Mao et al. 2005; Spitz et al. 2005), or in specific cell lineages (Zheng et al. 2000). The improvement of the chromosomal engineering technique is definitely based on the availability of a powerful Cre-expressing construct such as the Hprt1tm1(Cre)Mnn mouse line (Tang et al. 2002; Wu et al. 2007).
To go further into the analysis of the aneuploid mosaicism consequences, we used the property of the Cre recombinase to react on G2 phase of the cell cycle to develop a strategy for generating segmental aneuploid mosaicism in mouse tissues, allowing the study of the survival and the function of cells carrying microdeletion and microduplication. This strategy is based on a targeted asymmetric sister chromatid event of recombination (TASCER), due to a Cre-dependent recombination induced after the replication of DNA. In this article, we reveal for the first time the utility of TASCER to determine the consequences of segmental aneuploid mosaicism for two specific regions homologous to the telomeric part of HSA21 in the mouse.
MATERIALS AND METHODS
Gene targeting and mouse lines:
The targeting vectors for Prmt2 and U2af1 were isolated from the 3′HPRT library, whereas the Col6a1, Cstb, and Abcg1 targeting vectors were obtained from the 5′HPRT library (Zheng et al. 1999). LoxP sites were inserted in the same relative orientation by two successive homologous recombination events in HM-1 embryonic stem (ES) cells, as described in (Besson et al. 2007). For each targeting event, ES clones were selected for homologous recombination at the defined loci and injected into blastocysts to obtain chimeras. By crossing with C57BL/6J (B6) mice, we established the Cis(Prmt2-Col6a1)tm1Yah, Cis(Prmt2-Cstb)tm1Yah, and Cis(Abcg1-U2af1)tm1Yah mouse lines called Cis1 (Besson et al. 2007), Cis2, and Cis3, respectively, which carry the two loxP sites in a Cis configuration on Mus musculus (MMU) chromosome 10 (MMU10) for the first two lines and 17 (MMU17) for the last one.
Four transgenic mouse lines, expressing the CRE recombinase were used. They are either transgenic or site-targeted derived and express the Cre gene from various promoters: ubiquitously [Tg(CMV-Cre)1Pcn] (Dupe et al. 1997), during female ovogenesis [Tg(Zp3-Cre)93Knw](De Vries et al. 2000), in specific tissues, such as neural progenitors [Tg(Nes-Cre)1Kln] (Tronche et al. 1999), or in specific cell types from the myeloid lineage [Tg(MLys1-Cre)Cgn] (Clausen et al. 1999). Cre mouse lines were crossed with the “Cis” lines described above to generate double heterozygote individuals, which were studied further. All animals were bred under specific pathogen-free (SPF) conditions and were treated in compliance with animal welfare policies from the French Ministry of Agriculture (law 87 848 and YH accreditation 45-31).
Genotyping and sequencing:
Genomic DNA was extracted from tails, ES cells, and mouse organs, including testis, intestine, liver, kidney, muscles (gastrocnemius and biceps brachialis), heart, lung, brain, cerebellum, tongue, eyes, salivary glands, spleen, skin, stomach, and thymus. The analysis was carried out by PCR or Southern blot using standard operating procedures (Besson et al. 2007). Specific primer pairs (available upon request) were used to generate PCR-derived probes for the ampicillin and the neomycin resistance genes included in the targeting vectors.
In addition, PCR was performed to amplify specific 2-kb and 3-kb DNA fragments from the chromosomes carrying the deletion and the segmental duplication, respectively. To check the integrity of the recombined sequences, sequencing was performed with the BigDye Terminator V3.1 chemistry and analyzed with an ABI PRISM 310 apparatus (Applied Biosystem, Foster City, CA), as described previously (Besson et al. 2005).
Fluorescence in situ hybridization:
Interphase nuclei were recovered by affixing a sample of frozen/defrosted organs onto a slide. The mouse BACs were chosen from the RPCI-24 Mouse BAC Library (C57BL/6J Male, http://bacpac.chori.org/mmouse24.htm). BAC DNAs were purified with Nucleobond BAC 100 (Macherey-Nalgen, Hoerdt, France). One microgram of mouse BAC located inside (RPCI24-445L22) and outside (RPCI24-247G13) of the deleted region was used to generate DNA probes labeled by nick translation with DIG-dUTP (for 445L22) and biotin-dUTP (for 247G13) using ROCHE nick translation mix for in situ probe. The detection was realized by the use of both antidigoxigenin-rhodamine antibody and avidin-fluorescein (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instruction manual. The slides were mounted and images were captured as described previously (Besson et al. 2007).
RESULTS
Generating targeted asymmetric sister chromatid event of recombination:
To generate segmental mosaic aneuploidies on the two distinct chromosomes MMU10 (Cis1 and Cis2; Figure 1Figure 1. ) and MMU17 (Cis3; Figure 1Figure 1. ), we used several Cre-expressing transgenes. As a control experiment, we checked whether we were able to create the corresponding deletion from the Cis configuration (Cis/+). We crossed the Cis/+ mice with a general Cre deleter line, the Tg(CMV-Cre)1Pcn (Dupe et al. 1997). We were successful in generating the deletion Del1 of a 0.5-Mb genetic interval, as reported in 30% of newborns (n = 18) obtained by breeding the Cis1/+, Tg(CMV-Cre)1Pcn/0 mosaic mice, which carry the Cis configuration plus the deletion of the targeted region, with wild-type mice (Besson et al. 2007). Similarly, we tried with the Cis2 configuration, in which the two loxP sites are located 2.2 Mb apart. However, analysis of 183 progeny revealed that we did not get the transmission of the Del2 chromosomes from a transheterozygous animal carrying the Cis2/+, Tg(CMV-Cre)1Pcn/0. Nevertheless, we succeeded in vivo to recover Del2 using the Tg(Zp3-Cre)93Knw transgene in 3 of 83 animals. Furthermore, we detected the deleted band from the tail of such a Cis2/+, Tg(CMV-Cre)1Pcn/0 animal using a specific PCR assay (data not shown). At this point, we investigated whether the transheterozygous animal carrying the Cis2/+ chromosome and the Tg(CMV-Cre)1Pcn transgene was mosaic for the partial deletion of the corresponding region.
Figure 1.
Figure 1.
Figure 1.—
Schematic of the different genetic configurations used in the study located on mouse chromosome 10 (A) or 17 (B). (A) The loxP sites were inserted in a Cis configuration by gene targeting in ES cells in the Prmt2 and Col6a1 loci (Cis1). The Cre expression (more ...)
The CMV promoter is known to be expressed ubiquitously and early during embryogenesis (Zakany and Duboule 1996; Dupe et al. 1997; Spitz et al. 2005). Consequently, the Cre recombinase should be active in a variety of cell types using the Tg(CMV-Cre)1Pcn transgene (Dupe et al. 1997). We isolated DNA from 18 different organs from ~20 Cis2/+, Tg(CMV-Cre)1Pcn/0 mice to detect the corresponding Del2 deletion. Southern blot analysis, as shown in Figure 2AFigure 2. , unraveled a more complex situation than initially expected. The parental Cis2 alleles were detected in all organs (Figure 2AFigure 2. ). Surprisingly, additional bands could be associated with the presence of both the deletion (Del2) and the tandem duplication (Dup2) in a variety of organs, such as the kidney, different muscles, the tongue, the lung, or the cerebellum. The identification of the Dup2 was confirmed on the same blot by using a neomycin-specific probe that revealed the expected restriction pattern of the Dup2, the recombinant Cstb locus, and the integrated Cre transgene (Figure 2BFigure 2. ).
Figure 2.
Figure 2.
Figure 2.—
Detection of TASCER. (A–C) Southern blot analysis of targeted asymmetric chromatid event of recombination for the Prmt2-Ctsb and Prmt2-Col6a1 genetic intervals located on MMU10. Lanes 1–18 are DNA extracted from various tissues of one (more ...)
To produce both the deleted and the duplicated configurations, there should be a TASCER. This event takes place between the two loxP sites placed in a Cis configuration after the replication of cellular DNA when loxP are present on both sister chromatids (Figure 3Figure 3. ). A direct consequence of such a TASCER event is the generation of cells carrying partial monosomy and trisomy in a diploid context. Thus, to check this hypothesis, we performed a fluorescence in situ hybridization (FISH) analysis on nuclei isolated from gastrocnemius muscles of mutant animals, using a BAC probe located in the Prmt2-Cstb genetic interval and a control one outside the region (Figures 1Figure 1. and and2D).2DFigure 2. ). The number of detected trisomic (Ts) and monosomic (Ms) aneuploid nuclei for the Prmt2-Cstb interval in this tissue was relatively stable between the four independent individuals and reached 46.5 ± 6.4% of the observed nuclei (Table 1). Similar aneuploid mosaicism was observed with the Prmt2-Col6a1 interval, which is a MMU10 Prmt2-Cstb subregion (Figure 1Figure 1. ). The combination of the Cis1 allele with the Cre transgene leads to an equivalent distribution of Del1, the corresponding deletion, and Dup1, the respective duplication in the same panel of organs (Figure 2CFigure 2. ). Further investigation using muscles isolated from four Cis2/+, Tg(CMV-Cre)1Pcn/0 animals, aged from 28 days postpartum (dpp) to 156 dpp, revealed a stable level of aneuploid cells. However, we noticed little variation with aging (data not shown). In addition, we tested the variability of the TASCER event in five different tissues and we estimated the percentage of aneuploid nuclei using FISH analysis. As shown in Table 2, the level of aneuploid cells was very low in the kidney (19.0 ± 6.3%), the lung (25.0 ± 2.7%), and the brain (18.9 ± 6.4%). In addition, the percentage of monosomic cells was higher in these tissues, as observed from the Southern blot analysis. Both segmental trisomic and monosomic nuclei were found in equal amounts within muscles (18.3 ± 3.8% and 28.2 ± 3.4%, respectively) and cerebellum (19.6 ± 3.5% and 21.0 ± 4.7%, respectively) at similar levels in both populations. Altogether, these data demonstrated that the aneuploid mosaicism induced by the Tg(CMV-Cre)1Pcn/0 is stable between individuals for a given tissue.
Figure 3.
Figure 3.
Figure 3.—
The targeted asymmetric sister chromatid event of recombination (TASCER). Schematic of the TASCER that should take place in the cells carrying a Cis configuration with two loxP sites. After the S phase the Cis configuration has been replicated and could (more ...)
TABLE 1
TABLE 1
Percentage of aneuploid nuclei detected in muscles (gastrocnemius) of four Cis2/+; Tg(CMV-Cre)Pcn/0 animals
TABLE 2
TABLE 2
Percentage of aneuploid nuclei found in different tissues derived from four Cis2/+; Tg(CMV-Cre)Pcn/0 animals (mean ± SD)
We next analyzed the border of the new loci by direct sequencing of specific PCR products generated from the duplicated and the deleted regions. No modification of the sequence was observed and we confirmed that the recombination takes place as expected (Figure 4Figure 4. ). However, this absence of modification in the loxP sequence cannot explain why the duplicated chromosome, carrying three loxP sites in tandem, is still found in some cells. On the contrary, we would have expected that the loxP sites were the target of the Cre recombinase leading to the previous Cis configuration and ultimately to the deletion with a unique loxP site. Despite the fact that it is impossible to have germline transmission of the rearranged chromosome from the Cis2/+, Tg(CMV-Cre)1Pcn/0, as described above, we were able to detect the new genetic configuration in a few somatic tissues. These results definitely show that the CMV promoter is able to drive the Cre recombinase expression in a tissue-specific manner with the ability to generate a deletion and a duplication of the targeted region of interest. Moreover, the recombination event seems to be as accurate in this experiment, using long-range loxP sites spacing, as in more classical Cis configurations.
Figure 4.
Figure 4.
Figure 4.—
Controlling the TASCER event. (A) Detection of recombinant alleles. A PCR fragment encompassing the deletion was amplified as a specific 1987-bp DNA fragment using primer a located in the 3′HRPT vector and primer b in the 5′HPRT vector. (more ...)
Thus, starting from a Cis configuration with two loxP sites in the same relative orientation, located either 0.5 or 2.2 Mb apart, the expression of the Cre does not produce solely a complete deletion, but a more complex situation with monosomic and trisomic cells due to an efficient recombination after the G2 phase. This event should result from a higher activity of the Cre recombinase in dividing cells, but raises the question whether the TASCER is dependent on the tissue or on the Cre-expressing transgene.
Tissue-specific aneuploid mosaicism:
Although the TASCER event occurs in tissues from different embryonic origins, such as muscle and cerebellum, the segmental aneuploid mosaicism shows a large variability between tissues. Some tissues, such as the cerebellum and muscle, sustain or maintain the aneuploid conditions, while in others the level of aneuploid cells is reduced. In addition, the duplicated configuration is normally unstable with three unmodified loxP sites (Holzenberger et al. 2000). Consequently, duplication should be observed only (1) if the expression or the activity of the Cre recombinase is reduced or (2) if the local and tissue-specific structure of the chromosome does not allow the Cre to act on the loxP sites. Definitely the level of the Cre recombinase should reach a level that allows the recombination to occur in the tissue. Nevertheless the variability of aneuploid cells between different tissues may be a direct consequence of the Cre expression level or may result from the lethal effect induced by the aneuploidy during cellular development. Indeed, it is known that complete monosomy 21 often leads to premature death of the patient. Thus, we explored further the potential of the TASCER strategy by comparison with classical deletion induced by the Cre. We used additional transgenic lines expressing constitutive Cre in specific tissues, such as neural progenitors [Tg(Nes-Cre)1Kln] (Tronche et al. 1999), in cell types derived from the myeloid lineage [Tg(MLys1-Cre)Cgn] (Clausen et al. 1999), and in another cell line carrying two loxP sites located 0.6 Mb apart on MMU17 (Cis3; Figure 1Figure 1. ). In a series of experiments we tested the induction of the partial monosomy and trisomy in cells from different tissues that were not affected or analyzed previously, such as the brain, bone marrow, and bone-marrow-derived macrophages (BMDM) in culture. As a result, we observed similar recombination events leading to Del3 and Dup3 in different parts of the brain of Cis3/+; Tg(Nes-Cre)1Kln/0 mice but not in tissues where the Cre is not expressed with this transgene, such as liver, kidney, and muscle. These results show that TASCER could be used for another region of the mouse genome on MMU17 (Figures 1Figure 1. and and5A).5AFigure 5. ). Similarly, TASCER events were detected in the subregions of the brain isolated from the Cis2/+; Tg(Nes-Cre)1Kln/0 animals (Figure 5AFigure 5. ). TASCER could be induced both on MMU10 and MMU17 during neural development in progenitor cells while the Nestin promoter is expressed (Tronche et al. 1999), thus TASCER could be induced in different genomic contexts and in most organs. Finally, we investigated whether TASCER could be used to generate monosomic or trisomic cells in vitro. We prepared BMDM from four independent individuals with the Cis1/+, Tg(MLys1-Cre)Cgn/0 genotype. The Tg(MLys1-Cre)Cgn/0 transgene is known to be preferentially expressed in neutrophils and macrophages in vivo. A rapid analysis of several hematopoietic tissues from the double mutants (Figure 5CFigure 5. ) pinpointed that a few monosomic and trisomic cells were present in the bone marrow and to a lesser extent in lung. However, these two populations were nearly absent from the tissue, except for a few cells carrying the Del1 in the thymus or the spleen. On the contrary, the specific Del1 and Dup1 bands were observed in vitro in BMDM prepared from such Cis1/+, Tg(MLys1-Cre)Cgn/0 animals (Figure 5CFigure 5. ). This series of experiments using two new Cre expressing transgenes clearly demonstrates that TASCER can be widely and specifically induced for different genomic regions depending upon the specificity of the Cre driver transgene. Furthermore, it definitely confirms that both aneuploid conditions are compatible with cellular viability at least in several tissues.
Figure 5.
Figure 5.
Figure 5.—
Various Cre-expressing transgenes can induce TASCER in vivo or ex vivo. Different Cis configurations (A, Cis 3; B, Cis2, and C, Cis1) were associated with tissue-specific Cre-expressing lines [A and B, Tg(Nes-Cre)1Kln; C, Tg(MLys1-Cre)Cgn]. (more ...)
DISCUSSION
In this article we report a strategy, called TASCER, for engineering segmental aneuploidy mosaicism in vivo. We show, on two distinct chromosomes and with three different Cre-expressing transgenes, how TASCER can efficiently recover cells with partial monosomy and trisomy with a relatively constant level, in a variety of tissues. We took advantage of the post-S phase-induced Cre recombination on loxP sites inserted on the same chromosome in a Cis configuration separated by up to 2.2 Mb. Such an event has not been previously reported, presumably because the Cre is very active on loxP sites that are located nearby, such as in most Cre/loxP applications. At this point, the duplicated configuration that contains three loxP sites may happen but should react again to get a Cis or a deletion and could not be observed. In our Cis configuration where the two loxP sites are located far away, the distances tend to decrease from the Cre-mediated recombination frequency (Liu et al. 1998; Zheng et al. 2000) somehow preserving the chromosome with a large duplication. Although duplications were commonly produced to obtain germline transmission of new configurations (Puech et al. 2000; Zheng et al. 2000), an in vivo generated duplication coming from a Cis configuration has not been published to date.
The Cre-mediated recombination event that takes place in between loxP sites after the S phase is crucial to the TASCER process. TASCER clearly depends on the capacity of the targeted cells to proliferate and on the ability of the Cre transgene to be expressed during proliferation. Results obtained with the three distinct Cis configurations tested in this report demonstrate that in the mouse, the Cre recombinase is much more active during the G2 phase than in G1, a phenomenon already described in vitro (Liu et al. 1998, 2002; Yu and Bradley 2001) and discussed in vivo (Zong et al. 2005). Nevertheless, the in vitro efficiency was so low that the restoration of a selectable marker, such as the Hprt minigene, is required to select the event (Ramirez-Solis et al. 1995; Adams et al. 2004). In our experiment, the rate of recombination in TASCER is far more efficient than the in vitro induction in embryonic stem cells (Liu et al. 2002). Somehow, the Cre recombinase is more active in vivo, presumably due to stable conditions of expression in vivo, after the replication of DNA, in the G2 phase, while the chromatin structure is changing to prepare the future mitosis (Liu et al. 1998; Wu et al. 2007). We could also assume that the in vivo condition is more permissive to support the aneuploid cells due to the variety of tissue conditions. The physical connection of the sister chromatids due to structure, such as the cohesin complex that is found until anaphase, should facilitate the induced event of mitotic recombination between DNA (Losada et al. 2002; Takata et al. 2007). In addition, the TASCER observation suggests that the Cre is preferentially active in vivo while cells are proliferating.
As reported in previous studies, the Cre recombinase activity is dose dependent and varies with the driving promoter. The human cytomegalovirus sequence is a powerful promoter that directs Cre expression in early embryogenesis. Thus it has been used to completely knock out genes of interest or to generate large rearrangements (Zakany and Duboule 1996; Spitz et al. 2005). Here, we were able to induce up to 2.2 Mb long chromosomal rearrangements with a set of three promoters driving the expression of the Cre recombinase. Remarkably, although the duplicated configuration should be unstable due to the presence of three loxP sites in the same orientation (Holzenberger et al. 2000), trisomic cells were found in a variety of organs showing that trisomy might be beneficial in some tissues. We tried without success to generate larger aneuploid mosaicisms with the Tg(CMV-Cre)1Pcn transgene for a region of ~9.2 Mb. The recombination of a larger genetic interval might require a stronger promoter such as the CAG promoter inserted in the Hprt locus of the Hprt1tm1(Cre)Mnn mouse line, which has been used successfully in a recent study (Tang et al. 2002; Wu et al. 2007). As a consequence, inducing mosaic conditions in an adult animal will require preliminary experiments to balance the size of the targeted region with the expression level of the Cre gene. In addition, TASCER should be taken into account when trying to generate deletions in vivo. Additional controls to check for the duplication event should be carried out to avoid interference in the analysis of conditional knockouts for large genes or chromosomal regions. Thus, the degree of mosaicism generated through TASCER could vary in different tissues depending on the Cre transgene and on the proliferative capacities of the different cell types of each tissue.
Chromosomal mosaicism has been observed in pathological conditions, such as mosaic trisomy 21 (Modi et al. 2003) or in cancer (Weaver and Cleveland 2007). In this report, we followed the impact of time on the survival of cells that are trisomic and monosomic for small regions homologous to HSA21 and we did not find any dramatic changes. Thus, the presence of trisomic and monosomic cells for the analyzed regions does not affect the cell survival. Due to the cellular organization of muscle cells, any trisomic or monosomic consequences should be compensated by the plurinucleated syncitia of the muscle fiber. On the contrary, in the cerebellum the consequence of aneuploidy on cell survival could only be compensated through noncell autonomous effect.
To date, little evidence supports a functional consequence of partial trisomy or copy number variation in nonpathological conditions, even though aneuploidies have been described in normal lineages, such as neurons (Iourov et al. 2006; Kingsbury et al. 2006; Muotri and Gage 2006; Yurov et al. 2007a,b, 2008) but one could expect to use TASCER to study such impact. More importantly, TASCER allows us to induce partial aneuploid conditions that will mimic the aneuploidy detected in cancer cells and to study further the consequence of loss or gain of copy number for chromosomal regions during tumorigenesis (Weaver and Cleveland 2007). For example, looking for tumor suppressor genes in a region identified as an aneuploidy-induced loss of heterozygosity in cancer cells could be done on a larger scale with TASCER compared to classical loss-of-function studies that need gene-by-gene inactivation or ES-cell-derived technology such as Micer (Adams et al. 2004). In conclusion, we believe that TASCER opens new perspectives to assess the consequence of aneuploid conditions on viability and to decipher the impact of copy number variation during cell fate determination and tumorigenesis.
Acknowledgments
We thank members of the laboratory for helpful discussion, in particular Veronique Brault and Kathryn Jane Mitchell, and also Karine Maillard, Elodie Desale, and Tamara Durand for taking care of the animals and members of the AnEUploidy consortium (http://www.aneuploidy.org). We are grateful to P. Chambon, B. Knowkles, R. Klein, and I. Forster, respectively, for the Tg(CMV-Cre)1Pcn, the Tg(Zp3-Cre)93Knw, the Tg(Nes-Cre)1Kln, and the Tg(MLys1-Cre)Cgn mouse lines. The “Cis” mutant mice are available for distribution through the EMMA network (European Mouse Mutant Archive, http://www.emmanet.org). This work was funded by grants from the National Centre for Scientific Research (CNRS), the Region Centre, the Conseil Général du Loiret, Fondation Jerome Lejeune, and the AnEUploidy project (LSHG-CT-2006-037627) supported by the European commission under FP6.
References
  • Adams, D. J., P. J. Biggs, T. Cox, R. Davies, L. van der Weyden et al., 2004. Mutagenic insertion and chromosome engineering resource (MICER). Nat. Genet. 36 867–871. [PubMed]
  • Aitman, T. J., R. Dong, T. J. Vyse, P. J. Norsworthy, M. D. Johnson et al., 2006. Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans. Nature 439 851–855. [PubMed]
  • Besson, V., V. Nalesso, A. Herpin, J. C. Bizot, N. Messaddeq et al., 2005. Training and aging modulate the loss-of-balance phenotype observed in a new ENU-induced allele of Otopetrin1. Biol. Cell 97 787–798. [PubMed]
  • Besson, V., V. Brault, A. Duchon, D. Togbe, J. C. Bizot et al., 2007. Modeling the monosomy for the telomeric part of human chromosome 21 reveals haploinsufficient genes modulating the inflammatory and airway responses. Hum. Mol. Genet. 16 2040–2052. [PubMed]
  • Brault, V., P. Pereira, A. Duchon and Y. Herault, 2006. Modeling chromosomes in mouse to explore the function of genes, genomic disorders, and chromosomal organization. PLoS Genet. 2 e86. [PubMed]
  • Cheung, S. W., C. A. Shaw, D. A. Scott, A. Patel, T. Sahoo et al., 2007. Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics. Am. J. Med. Genet. A 143 1679–1686.
  • Clausen, B. E., C. Burkhardt, W. Reith, R. Renkawitz and I. Forster, 1999. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 8 265–277. [PubMed]
  • de Vries, W. N., L. T. Binns, K. S. Fancher, J. Dean, R. Moore et al., 2000. Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis 26 110–112. [PubMed]
  • Dupe, V., M. Davenne, J. Brocard, P. Dolle, M. Mark et al., 1997. In vivo functional analysis of the Hoxa-1 3′ retinoic acid response element (3′RARE). Development 124 399–410. [PubMed]
  • Fellermann, K., D. E. Stange, E. Schaeffeler, H. Schmalzl, J. Wehkamp et al., 2006. A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon. Am. J. Hum. Genet. 79 439–448. [PubMed]
  • Feuk, L., A. R. Carson and S. W. Scherer, 2006. a Structural variation in the human genome. Nat Rev. Genet. 7 85–97. [PubMed]
  • Feuk, L., A. Kalervo, M. Lipsanen-Nyman, J. Skaug, K. Nakabayashi et al., 2006. b Absence of a paternally inherited FOXP2 gene in developmental verbal dyspraxia. Am. J. Hum. Genet. 79 965–972. [PubMed]
  • Freeman, J. L., G. H. Perry, L. Feuk, R. Redon, S. A. McCarroll et al., 2006. Copy number variation: new insights in genome diversity. Genome Res. 16 949–961. [PubMed]
  • Genoud, N., A. Behrens, G. Miele, D. Robay, F. L. Heppner et al., 2004. Disruption of Doppel prevents neurodegeneration in mice with extensive Prnp deletions. Proc. Natl. Acad. Sci. USA 101 4198–4203. [PubMed]
  • Gonzalez, E., H. Kulkarni, H. Bolivar, A. Mangano, R. Sanchez et al., 2005. The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility. Science 307 1434–1440. [PubMed]
  • Hahnemann, J. M., and L. O. Vejerslev, 1997. European collaborative research on mosaicism in CVS (EUCROMIC)–fetal and extrafetal cell lineages in 192 gestations with CVS mosaicism involving single autosomal trisomy. Am. J. Med. Genet. 70 179–187. [PubMed]
  • Hassold, T., and P. Hunt, 2001. To err (meiotically) is human: the genesis of human aneuploidy. Nat. Rev. Genet. 2 280–291. [PubMed]
  • Hassold, T., H. Hall and P. Hunt, 2007. The origin of human aneuploidy: where we have been, where we are going. Hum. Mol. Genet. 16(Spec No. 2): R203–R208. [PubMed]
  • Herault, Y., M. Rassoulzadegan, F. Cuzin and D. Duboule, 1998. Engineering chromosomes in mice through targeted meiotic recombination (TAMERE). Nat. Genet. 20 381–384. [PubMed]
  • Hollox, E. J., U. Huffmeier, P. L. Zeeuwen, R. Palla, J. Lascorz et al., 2008. Psoriasis is associated with increased beta-defensin genomic copy number. Nat. Genet. 40 23–25. [PubMed]
  • Holzenberger, M., C. Lenzner, P. Leneuve, R. Zaoui, G. Hamard et al., 2000. Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene. Nucleic Acids Res. 28 21.
  • Iourov, I. Y., S. G. Vorsanova and Y. B. Yurov, 2006. Chromosomal variation in mammalian neuronal cells: known facts and attractive hypotheses. Int. Rev. Cytol. 249 143–191. [PubMed]
  • Kalousek, D. K., 2000. Pathogenesis of chromosomal mosaicism and its effect on early human development. Am. J. Med. Genet. 91 39–45. [PubMed]
  • Katz-Jaffe, M. G., A. O. Trounson and D. S. Cram, 2004. Mitotic errors in chromosome 21 of human preimplantation embryos are associated with non-viability. Mol. Hum. Reprod. 10 143–147. [PubMed]
  • Katz-Jaffe, M. G., A. O. Trounson and D. S. Cram, 2005. Chromosome 21 mosaic human preimplantation embryos predominantly arise from diploid conceptions. Fertil. Steril. 84 634–643. [PubMed]
  • Kidd, J. M., G. M. Cooper, W. F. Donahue, H. S. Hayden, N. Sampas et al., 2008. Mapping and sequencing of structural variation from eight human genomes. Nature 453 56–64. [PubMed]
  • Kingsbury, M. A., B. Friedman, M. J. McConnell, S. K. Rehen, A. H. Yang et al., 2005. Aneuploid neurons are functionally active and integrated into brain circuitry. Proc. Natl. Acad. Sci. USA 102 6143–6147. [PubMed]
  • Kingsbury, M. A., Y. C. Yung, S. E. Peterson, J. W. Westra and J. Chun, 2006. Aneuploidy in the normal and diseased brain. Cell. Mol. Life Sci. 63 2626–2641. [PubMed]
  • Kmita, M., N. Fraudeau, Y. Herault and D. Duboule, 2002. Serial deletions and duplications suggest a mechanism for the collinearity of Hoxd genes in limbs. Nature 420 145–150. [PubMed]
  • Lewandoski, M., and G. R. Martin, 1997. Cre-mediated chromosome loss in mice. Nat. Genet. 17 223–225. [PubMed]
  • Liu, P., N. A. Jenkins and N. G. Copeland, 2002. Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells. Nat. Genet. 30 66–72. [PubMed]
  • Liu, P., H. Zhang, A. McLellan, H. Vogel and A. Bradley, 1998. Embryonic lethality and tumorigenesis caused by segmental aneuploidy on mouse chromosome 11. Genetics 150 1155–1168. [PubMed]
  • Losada, A., M. Hirano and T. Hirano, 2002. Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis. Genes Dev. 16 3004–3016. [PubMed]
  • Lupski, J. R., and P. Stankiewicz, 2005. Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoS Genet. 1 e49. [PubMed]
  • Mao, R., X. Wang, E. L. Spitznagel, Jr., L. P. Frelin, J. C. Ting et al., 2005. Primary and secondary transcriptional effects in the developing human Down syndrome brain and heart. Genome Biol. 6 R107. [PubMed]
  • Mikkelsen, M., 1977. Down syndrome: cytogenetical epidemiology. Hereditas 86 45–50. [PubMed]
  • Modi, D., P. Berde and D. Bhartiya, 2003. Down syndrome: a study of chromosomal mosaicism. Reprod. Biomed. Online 6 499–503. [PubMed]
  • Muotri, A. R., and F. H. Gage, 2006. Generation of neuronal variability and complexity. Nature 441 1087–1093. [PubMed]
  • Puech, A., B. Saint-Jore, S. Merscher, R. G. Russell, D. Cherif et al., 2000. Normal cardiovascular development in mice deficient for 16 genes in 550 kb of the velocardiofacial/DiGeorge syndrome region. Proc. Natl. Acad. Sci. USA 97 10090–10095. [PubMed]
  • Ramirez-Solis, R., P. Liu and A. Bradley, 1995. Chromosome engineering in mice. Nature 378 720–724. [PubMed]
  • Redon, R., S. Ishikawa, K. R. Fitch, L. Feuk, G. H. Perry et al., 2006. Global variation in copy number in the human genome. Nature 444 444–454. [PubMed]
  • Rehen, S. K., M. J. McConnell, D. Kaushal, M. A. Kingsbury, A. H. Yang et al., 2001. Chromosomal variation in neurons of the developing and adult mammalian nervous system. Proc. Natl. Acad. Sci. USA 98 13361–13366. [PubMed]
  • Rehen, S. K., Y. C. Yung, M. P. McCreight, D. Kaushal, A. H. Yang et al., 2005. Constitutional aneuploidy in the normal human brain. J. Neurosci. 25 2176–2180. [PubMed]
  • Robinson, W. P., F. Bernasconi, A. Lau and D. E. McFadden, 1999. Frequency of meiotic trisomy depends on involved chromosome and mode of ascertainment. Am. J. Med. Genet. 84 34–42. [PubMed]
  • Robinson, W. P., D. E. McFadden and M. D. Stephenson, 2001. The origin of abnormalities in recurrent aneuploidy/polyploidy. Am. J. Hum. Genet. 69 1245–1254. [PubMed]
  • Sebat, J., B. Lakshmi, D. Malhotra, J. Troge, C. Lese-Martin et al., 2007. Strong association of de novo copy number mutations with autism. Science 316 445–449. [PubMed]
  • Sharp, A. J., R. R. Selzer, J. A. Veltman, S. Gimelli, G. Gimelli et al., 2007. Characterization of a recurrent 15q24 microdeletion syndrome. Hum. Mol. Genet. 16 567–572. [PubMed]
  • Sharp, A. J., H. C. Mefford, K. Li, C. Baker, C. Skinner et al., 2008. A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures. Nat. Genet. 40 322–328. [PubMed]
  • Shaw, C. J., and J. R. Lupski, 2004. Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease. Hum. Mol. Genet. 13(Spec No. 1): R57–R64. [PubMed]
  • Spitz, F., C. Herkenne, M. A. Morris and D. Duboule, 2005. Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes. Nat. Genet. 37 889–893. [PubMed]
  • Takata, H., S. Matsunaga, A. Morimoto, N. Ma, D. Kurihara et al., 2007. PHB2 protects sister-chromatid cohesion in mitosis. Curr. Biol. 17 1356–1361. [PubMed]
  • Tang, S. H., F. J. Silva, W. M. Tsark and J. R. Mann, 2002. A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ. Genesis 32 199–202. [PubMed]
  • Tarchini, B., and D. Duboule, 2006. Control of Hoxd genes' collinearity during early limb development. Dev. Cell 10 93–103. [PubMed]
  • Tronche, F., C. Kellendonk, O. Kretz, P. Gass, K. Anlag et al., 1999. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Genet. 23 99–103. [PubMed]
  • Weaver, B. A., and D. W. Cleveland, 2007. Aneuploidy: instigator and inhibitor of tumorigenesis. Cancer Res. 67 10103–10105. [PubMed]
  • Wu, S., G. Ying, Q. Wu and M. R. Capecchi, 2007. Toward simpler and faster genome-wide mutagenesis in mice. Nat. Genet. 39 922–930. [PubMed]
  • Yu, Y., and A. Bradley, 2001. Engineering chromosomal rearrangements in mice. Nat. Rev. Genet. 2 780–790. [PubMed]
  • Yurov, Y. B., I. Y. Iourov, S. G. Vorsanova, T. Liehr, A. D. Kolotii et al., 2007. a Aneuploidy and confined chromosomal mosaicism in the developing human brain. PLoS ONE 2 e558. [PubMed]
  • Yurov, Y. B., S. G. Vorsanova, I. Y. Iourov, I. A. Demidova, A. K. Beresheva et al., 2007. b Unexplained autism is frequently associated with low-level mosaic aneuploidy. J. Med. Genet. 44 521–525. [PubMed]
  • Yurov, Y. B., I. Y. Iourov, S. G. Vorsanova, I. A. Demidova, V. S. Kravetz et al., 2008. The schizophrenia brain exhibits low-level aneuploidy involving chromosome1. Schizophr. Res. 98 139–147. [PubMed]
  • Zakany, J., and D. Duboule, 1996. Synpolydactyly in mice with a targeted deficiency in the HoxD complex. Nature 384 69–71. [PubMed]
  • Zakany, J., M. Kmita and D. Duboule, 2004. A dual role for Hox genes in limb anterior-posterior asymmetry. Science 304 1669–1672. [PubMed]
  • Zheng, B., A. A. Mills and A. Bradley, 1999. A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice. Nucleic Acids Res. 27 2354–2360. [PubMed]
  • Zheng, B., M. Sage, E. A. Sheppeard, V. Jurecic and A. Bradley, 2000. Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol. Cell. Biol. 20 648–655. [PubMed]
  • Zong, H., J. S. Espinosa, H. H. Su, M. D. Muzumdar and L. Luo, 2005. Mosaic analysis with double markers in mice. Cell 121 479–492. [PubMed]
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