Mouse chromosome identification - Fig. 1

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Figure legend:

Fig. 1. A metaphase image from a transgenic female mouse, after hybridization with the triple-color labeled panel of BACs (i) or after DAPI staining (ii). Individual chromosomes from this spread are separately displayed (iii) to reflect the labeling scheme (iv). Three fluorophores were used to label these probes: FITC (green 4 G), R6G (red 4 R), and AMCA (blue 4 B). Three test probes labeled with the RB combination (magenta) are mapped on this metaphase. Two of the test probes (BACs) hybridize to locations A (Chr 14) and B (Chr 16). A multicopy transgene, integrated in the major satellite of Chr 5, was probed with the transgene construct and is shown as C in image (v). Although probes B and C are each close to a reference probe, the coloring algorithm allows specific identification of the test probe. Simultaneous use of more than three colors allows numerous probes to be mapped simultaneously (vi-vii). We labeled probe A with FITC-dUTP and dinitrophenol (DNP)-dUTP, probe B with digoxigenin-dUTP, and probe D with CY3-dUTP (detected through the same filter as R6G) and DNP-dUTP. DNP was detected with an antibody labeled with CY3.5, and DIG with a DEAC-labeled antibody. The positions of probes A, B, and D are shown along with the reference probe panel in the FISH image (vi), and separately on the inverted DAPI image (vii). This indicates that multiple unknown probes can be mapped in a single hybridization with this technique. Mouse splenocyte cultures were grown for 48 h with con-canavalin A and b-mercaptoethanol, after which the cells were resuspended in 20 ml of 75 mM KCl for 10 min, at which time 1/40 volume of fixative (3:1 methanol:acetic acid) was added. The cells were pelleted and then washed four times in the fixative (Alami et al. 2000). Slides were prepared as described (Henegariu et al. 1999) and stored at •20°C in 100% ethanol. BAC clones (Korenberg et al. 1999) are available from Research Genetics (Huntsville, Ala.) for US$47.00 per clone (catalog number MB11301). BAC DNA was prepared with commercial alkaline lysis kits labeled by the degenerate oligonucleotide priming polymerase chain reaction (DOP-PCR), with the previously described primers A and B (Bohlander et al. 1992). In the first PCR reaction, 50-100 ng DNA of each probe was amplified by DOP-PCR in separate vials. The PCR program used was: 2 cycles of denaturation at 94°C (45 s), annealing at 15°C (40 s), and extension at 37°C (10 min); 5 cycles of denaturation at 94°C (45 s), annealing at 37°C (40 s), and extension at 66°C (3-4 min); 25 cycles of denaturation at 94°C (45 s), annealing at 54°C (40 s), and extension at 66°C (3-4 min). The product of these reactions was used as template for the subsequent labeling PCR reactions. PCR labeling was performed in 12 different vials, combining 2-3 ml of DNA template from each of 4-5 probes to be labeled with the same flour, in one vial (100-ml reactions). The PCR program was identical to the last part of the program detailed above, but was run for 30 cycles, not 25. Custom-made nucleotides were used to label the DNA and were used as detailed elsewhere (Henegariu et al. 2000). One round of PCR labeling generates sufficient probe for up to six hybridizations. For each hybridization, the equivalent of 4-5ml of labeled DNA for each probe (for a total of about 200 ml total PCR product) were combined in the same vial, cleaned, and adjusted to 200-300bp average length as described (Henegariu et al. 2000). The DNA was combined with 70 mg of mouse Cot-1 DNA, ethanol precipitated, resus-pended in 12 ml of hybridization buffer (50% formamide/10% dextran sulfate/2x SSC) and hybridized. A thorough discussion of the various slide preparation procedures and hybridization protocols is detailed elsewhere (Henegariu et al. 2001). The slides were viewed using a fluorescence microscope (Olympus AX-70) equipped with a cooled CCD camera (Pho-tometrics) and appropriate filters (Henegariu et al. 2000). Images were processed with Photoshop (Adobe), by merging and pseudocoloring the black-and-white images for each fluorescent dye.