Figure 1.
Figure 1. Chromosomal translocations between nonhomologous sequences. (A) Translocation design. A neo gene is split within its intron such that the 5′ portion with the splice donor (neoSD) is targeted to chromosome 17 in mouse ES cells, and the 3′ portion with the splice acceptor (SAneo) is targeted to chromosome 14. DSB induction at the I-SceI sites followed by interchromosomal NHEJ results in a neo+ gene on der(17). Because the breakpoint junction occurs within an intron, a variety of junctions can be recovered. The reciprocal chromosome der(14) also can form but is not under selection. Gray box, selectable marker. (B) Translocation reporters p5rE and p5rErev. The 2 rE alleles differ by whether the I-SceI sites on chromosomes 17 and 14 are in the same (rE) or opposite orientation (rErev). Der(17) arising from NHEJ with minimal sequence alteration reconstructs a neo+ gene with an intron of approximately 1.7 kb; der(14) can arise from NHEJ or SSA at the 210-bp repeat (green box), as shown. In principle, homology-directed repair at the 210-bp repeat could cause translocation formation via a reciprocal exchange. However, consistent with previous results,9-12 homology-directed repair events, which would leave one copy of the repeat on der(17), have not been recovered; hence, these translocation chromosomes are not diagrammed. Key distances are indicated in kb. (C) Translocation reporter pCr15. Der(17) arising from NHEJ with minimal sequence alteration reconstructs a neo+ gene with an intron of approximately 2.3 kb; like der(17), der(14) also can only arise from NHEJ. The I-SceI site is positioned further from the splice donor in the pCr15 reporter than in the p5rE and p5rErev reporters. For the pCr15 reporter, the I-SceI sites are in opposite orientation. The sequence of the I-SceI site upon cleavage is shown (blue box). (D) Fluorescence in situ hybridization using whole mouse chromosome 14-FITC (green) and chromosome 17-Cy3 (red) probes and demonstrating normal chromosomes 14 and 17 in the parental cell line and 2 derivative chromosomes (yellow arrows) in a neo+ clone. (E) Translocation chromosome analysis. PCR primers and sizes of PCR products are indicated on the derivative chromosomes in panel B. A sample of PCR products obtained from p5rE neo+ clones shows der(17) NHEJ junctions of variable length and der(14) NHEJ and SSA products (1.7 and 0.9 kb, respectively). No band is obtained from the parental cell line since the primers are located on different chromosomes.

Chromosomal translocations between nonhomologous sequences. (A) Translocation design. A neo gene is split within its intron such that the 5′ portion with the splice donor (neoSD) is targeted to chromosome 17 in mouse ES cells, and the 3′ portion with the splice acceptor (SAneo) is targeted to chromosome 14. DSB induction at the I-SceI sites followed by interchromosomal NHEJ results in a neo+ gene on der(17). Because the breakpoint junction occurs within an intron, a variety of junctions can be recovered. The reciprocal chromosome der(14) also can form but is not under selection. Gray box, selectable marker. (B) Translocation reporters p5rE and p5rErev. The 2 rE alleles differ by whether the I-SceI sites on chromosomes 17 and 14 are in the same (rE) or opposite orientation (rErev). Der(17) arising from NHEJ with minimal sequence alteration reconstructs a neo+ gene with an intron of approximately 1.7 kb; der(14) can arise from NHEJ or SSA at the 210-bp repeat (green box), as shown. In principle, homology-directed repair at the 210-bp repeat could cause translocation formation via a reciprocal exchange. However, consistent with previous results,9-12  homology-directed repair events, which would leave one copy of the repeat on der(17), have not been recovered; hence, these translocation chromosomes are not diagrammed. Key distances are indicated in kb. (C) Translocation reporter pCr15. Der(17) arising from NHEJ with minimal sequence alteration reconstructs a neo+ gene with an intron of approximately 2.3 kb; like der(17), der(14) also can only arise from NHEJ. The I-SceI site is positioned further from the splice donor in the pCr15 reporter than in the p5rE and p5rErev reporters. For the pCr15 reporter, the I-SceI sites are in opposite orientation. The sequence of the I-SceI site upon cleavage is shown (blue box). (D) Fluorescence in situ hybridization using whole mouse chromosome 14-FITC (green) and chromosome 17-Cy3 (red) probes and demonstrating normal chromosomes 14 and 17 in the parental cell line and 2 derivative chromosomes (yellow arrows) in a neo+ clone. (E) Translocation chromosome analysis. PCR primers and sizes of PCR products are indicated on the derivative chromosomes in panel B. A sample of PCR products obtained from p5rE neo+ clones shows der(17) NHEJ junctions of variable length and der(14) NHEJ and SSA products (1.7 and 0.9 kb, respectively). No band is obtained from the parental cell line since the primers are located on different chromosomes.

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