Inducing segmental aneuploid mosaicism in the mouse through targeted asymmetric sister chromatid event of recombination

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.

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 generates the corresponding deletion (Del1) and duplication (Dup1). Cis2 corresponds to the insertion of a loxP site in a Cis configuration in the Prmt2 and Cstb loci while Del2 and Dup2 are the respective deletion and duplication of the 2.2-Mb-long genetic interval. The localization of the BAC used for the FISH analysis is shown (orange). (B) An additional Cis configuration (Cis3) with the corresponding deletion and duplication, respectively Del3 and Dup3, was used in the study on MMU17 with two loxP sites targeted at the level of the Abcg1 and U2af1 genes. The positions of the targeting vectors are shown in blue with the loxP sites represented as a green arrow. The size of the restriction bands identified by probe A (red box) or probe B (black box) are indicated in kilobases. Bg, BglII; H, HindIII; E, EcoRI.

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 tested male carrying the Cis2 (A and B) or the Cis1 (C) alleles and hybridized with the Amp (A and C) or Neo (B) probes, as indicated. (A) Bands corresponding to the bordering locus of Cis2 are found in all the lanes. No Cre-dependent recombination is induced in the testis, brain, or stomach, whereas the deletion Del2 is observed in the intestine, tail, and salivary gland. Moreover, balanced Dup2 and Del2 are detected in the kidney, muscles (gastrocnemius and biceps brachialis), lung, cerebellum, tongue, eyes, and skin. Accordingly, tissues where the balanced Del2 and Dup2 were detected (A) displayed a specific band for the duplication when hybridized with the Neo probe. The Neo probe hybridized also with the Cstb locus and the Cre transgene in all the lanes (B). (C) Similar tissues were isolated from a Cis1/+, Tg(CMV-Cre)Pcn/0 individual and bands corresponding to the Del1 and the Dup1 were detected in the same panel of tissues (Del1: lanes 2–8, 10, 11, 13–17; Dup1: lanes 3–8, 10, 11, 16, 17). (D) Interphase FISH analysis with BAC probes that map in the region of the deletion and duplication (red). The wild-type (2n) nuclei showed two red signals whereas those carrying the Del2 or the Dup2 displayed, respectively, one or three red signals for the Prmt2-Cstb region.

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 be the target of the Cre recombinase in an asymmetric way leading to monosomic and trisomic daughter cells after mitosis.

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. For the duplication, a PCR was performed to amplify a specific 3343-bp fragment using primer d located in the puromycin-resistant gene and primer e located in the neomycin-resistant gene. (B) PCR amplification for deletion using genomic DNA isolated from the muscle of a mosaic animal, using both primers (a + b). The fragments (a + b) were purified and sequenced using primers a, b, and c. (C) PCR amplification for duplication using the same animal tissue with both primers d and e. The fragment (d + e) was purified and sequenced using primers d, e, and c. (D) DNA sequence of fragment amplified in B and C with primer c. Color of the sister chromatid DNA source is similar to A. Restriction sites are underlined and the sequence of the loxP sites is shown in green.

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]. (A) Southern blot analysis of DNA isolated from the tissues of Cis3/+, Tg(Nes-Cre)1Kln/0 animals that do not express the Cre recombinase (lanes 1, 2, and 3: liver, kidney, and muscle) or are expressing the Cre (different parts of the brain: lanes 4–9) revealed with the Amp probes and different restriction enzymes (top, EcoRI; bottom, HindIII), the bordering loci of the Cis3 regions (Abcg1 and U2af1), and the recombined fragment either Del3 (bottom, lanes 4–9) or Dup3 (top, lanes 4–9) resulting from TASCER. (B) Similar results were obtained during the analysis of several subparts of the brain [hippocampus (1), olfactory bulbs (2), cortex (3), medulla oblongata (4), colliculus (5), thalamus (6), cerebellum (7)] of a Cis2/+, Tg(Nes-Cre)1Kln/0 individual showing that TASCER was induced efficiently in the different parts of the brain with the Dup2 and Del2 specific bands. (C) The Tg(MLys1-Cre)Cgn expressing line in macrophages and neutrophils was combined with the Cis1 locus. Specific bands for Cis1, Del1, and Dup1 were strongly detected ex vivo in bone-marrow-derived macrophages (BMDM: 5, 6, and 7) and in bone marrow (lane 4) whereas the recombination seems to be less efficient in the lung (lane 1), thymus (lane 2), and spleen (lane 3) from Cis1/+, Tg(MLys1-Cre)Cgn animals.