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A chromosome engineering strategy that allows the generation of chromosomes with this …


Biology Articles » Bioengineering » Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing » Figures

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- Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing

mcith_ddi31401.gif Figure 1 Schematic diagram of the Cre/loxP chromosome engineering strategy. A diagram of the mouse genomic Aprt gene, with a unique HindIII site in intron 2, is shown. The 5' portion of the Aprt gene was separated from the 3' portion at this unique HindIII site. A floxed Neo or Hyg resistance gene was inserted at the HindIII site in both the 5' and 3' portions of the Aprt gene, respectively, resulting in the 5'-AP-Neo and Hyg-3'RT cassettes, as shown. The 5'-AP-Neo and Hyg-3'RT cassettes integrate randomly throughout the genome following linearization and electroporation. After Cre transient transfection, reciprocal translocations are generated in a two-step process. First, due to the close proximity of the loxP sites flanking the Neo and Hyg genes, and the fact that they are aligned in the same orientation, the Neo and Hyg genes are excised as circles via highly efficient (determined to be nearly 90%; data not shown) intra-chromosomal events. Next, Cre directs the remaining loxP sites to proceed through a low efficiency (<1x10–3) inter-chromosomal reciprocal exchange. This results in reconstitution of the Aprt gene on one derivative chromosome, and a single loxP site on the other derivative, converting cells from APRT (P-clones) to APRT+ (R-pools). A preliminary description of this system was described elsewhere (9).

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mcith_ddi31402.gif Figure 2 Representative examples of DMC from the initial screen. Following Cre transient transfection and pooling of Aprt+ colonies, 30–50 metaphase spreads from each R-pool were scored for the presence of chromosomes with DMC. Examples of DMC from R27A (AC), R186A (DF), R230A (G, H) and R268A (I) are shown. Arrows mark chromosomes with DMC. Chromosomes were stained with propidium iodide (PI).

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mcith_ddi31403.gif Figure 3 Cytogenetic characterization of P175 and R175. (A) FISH on P175 using the plasmids containing the 5'-AP-Neo and Hyg-3'RT cassettes as probes. Arrows mark the chromosome 10 and 6 insertion sites on a representative mitotic spread. Chromosomes were stained with propidium iodide (PI). (B) R-banding of the mitotic spread shown in (A). Arrows mark the chromosome 10 and 6 insertion sites and are: 6q12–13 and 10q11.2. (C) FISH on a representative mitotic spread from R175A using the plasmids containing the 5'-AP-Neo and Hyg-3'RT constructs as probes. R-banding indicated that a balanced t(6;10)(q12–13;q11.2) was generated at the plasmid cassette insertion sites (not shown). Arrows mark the chromosome 10 (der10) and 6 (der6) derivatives. Chromosomes were stained with propidium iodide (PI). (D) FISH using chromosome 6 (red) and 10 (green) WCPs on a representative mitotic spread from R175A. Arrows mark the balanced t(6;10). Chromosomes were stained with DAPI. (E) Representative G-banded chromosomes from P175 and R175A (R175) showing the normal chromosomes 6 and 10 in P175 and the t(6;10) in R175A. (F) A schematic representation of the plasmid insertion sites in P175 and the Cre-dependent t(6;10) generated in R175. A similar analysis of an independent R175 pool (R175B) indicated that the same balanced t(6;10) was generated (not shown).

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mcith_ddi31404.gif Figure 4 DMC on the Cre-dependent t(6;10) in R175. (A) FISH using a chromosome 6 WCP as probe on a mitotic spread from R175A. The arrow marks a chromosome with DMC that partially hybridizes to the WCP. (B) FISH using a chromosome 10 WCP as probe on a mitotic spread from R175A. The arrow marks a chromosome with DMC that partially hybridized to the WCP. The inset shows the DAPI image of the delayed chromosome. (C, D) FISH, using both chromosome 6 (red) and 10 (green) WCPs, on a mitotic spread from R175B. The arrows mark a chromosome with DMC that hybridizes to both WCPs. (E, F) FISH, using chromosome 6- (red) and 10- (green) specific centromeric probes, on mitotic spreads from R175B. Arrows mark chromosomes with DMC. A similar analysis on R175A indicated that the chromosome 10, not the chromosome 6 derivative, displayed DMC (not shown). The chromosomes were stained with DAPI.

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mcith_ddi31405.gif Figure 5 Cytogenetic analysis of P186 and R186. (A) FISH, using the Aprt plasmid (P) cassettes, and R-banding (R) on chromosomes from P186. Hybridization was detected at 3q29, 13q14 and 11p15. (B) G-banding of the chromosomes involved in the Cre-dependent t(3;13) present in R186A. A similar analysis of R186B indicated that the same t(3;13) was generated (not shown). (C) A schematic representation of the Cre-dependent t(3;13) generated in R186. (D, E) DMC of the Cre-dependent t(3;13) in R186A. (D) A representative mitotic spread from R186A. (E) FISH using a chromosome 3 WCP as probe on the mitotic spread from panel D. The arrows mark chromosomes with DMC that hybridize to the chromosome 3 WCP, the asterisks (*) mark non-rearranged chromosome 3s, the arrowhead marks the pre-existing t(3;11) (Supplementary Material, Fig. S1), the pluses (+) mark unexpected translocations involving chromosome 3 (see below) and the double arrows mark the chromosome 13 derivative from the t(3;13). (F, G) FISH, using chromosome 3- (green) and chromosome 13- (red) specific centromeric probes, on two mitotic spreads from R186A. Arrows mark chromosomes with DMC that hybridize to either the chromosome 3 (F) or chromosome 13 (G) centromeric probes. Chromosomes were stained with DAPI.

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mcith_ddi31406.gif Figure 6 Engineered chromosomes with DMC also have DRT. (A) A schematic representation of the replication timing assay. The average duration of the G2 phase for R175B was 4–5 h (not shown). R175B cells were pulsed with BrdU for 15 min and mitotic harvests were prepared at 3-, 4- and 5-h time points. (BD) FISH, using a chromosome 10-specific centromeric probe (red) combined with immunostaining with an anti-BrdU antibody (green), on a mitotic spread from R175B pulsed 4 h earlier with BrdU. Note that all of the normally condensed chromosomes showed no BrdU incorporation and that the chromosome 10 derivative (arrow) showed BrdU incorporation along the entire length of the chromosome.

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mcith_ddi31407.gif Figure 7 Cytogenetic analysis of RS175-1. (A, B) The t(6;10) generated from P175 following expression of I-Sce1 is cytogenetically identical to t(6;10) generated following expression of Cre. FISH, using chromosome 6- (red) and 10- (green) specific WCPs, on a mitotic spread harvested with a colcemid pre-treatment step from RS175-1 (B). R-banding of mitotic spreads following the WCPs indicated the t(6;10) was indistinguishable from the t(6;10)(q12–13;q11.2) generated in R175A and R175B. Chromosomes were stained with DAPI, and arrows mark the t(6;10). (C) A schematic representation of the t(6;10) generated following expression of I-Sce1 in P175. (D) DMC on the chromosome 10 derivative in RS175-1. FISH, using chromosome 6- (red) and 10- (green) specific centromeric probes, on a mitotic spread prepared in the absence of a colcemid pre-treatment step from RS175-1. The arrow marks a chromosome with DMC that hybridized to the chromosome 10 centromeric probe.

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mcith_ddi31408.gif Figure 8 Chromosomes with DRT/DMC are unstable. Examples of FISH using WCPs as probes on cells from independent clones either before (P244F and P268F) or after Cre (R244F and R268F). All mitotic spreads were prepared in the presence of a colcemid pre-treatment step. (A) P244 was transfected with an Aprt-expression vector, selected in AA media, and individual clones (P244F1–5) were isolated and expanded through 20–22 cell doublings. Mitotic spreads were hybridized simultaneously to chromosome 13 (red) and 16 (green) WCPs. (B) P244 was transfected with a Cre-expression vector, selected in AA containing media, and individual clones (R244F1–5) were isolated and expanded through 20–22 cell doublings. Mitotic spreads were hybridized simultaneously with chromosome 13 (red) and 16 (green) WCPs. (C) P268 was transfected with an Aprt-expression vector, selected in AA media, and individual clones (P268F1–4) were isolated and expanded through 20–22 cell doublings. Mitotic spreads were hybridized simultaneously to chromosome 15 (green) and 16 (red) WCPs. (D) P268 was transfected with a Cre-expression vector, selected in AA media, and individual clones (R268F1–5) were isolated and expanded through 20–22 cell doublings. Mitotic spreads were hybridized simultaneously to chromosome 15 (red) and 16 (green) WCPs. One hundred mitotic spreads were analyzed for each clone. Each panel shows representative FISH+ chromosomes from each clone.

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