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Figure 1 | BMC Research Notes

Figure 1

From: Heat-induced and spontaneous expression of Hsp70.1Luciferase transgene copies localized on Xp22 in female bovine cells

Figure 1

The luciferase transgenes are located on bovine Xp22 and do not alter random X-chromosome inactivation. (A) A whole chromosome metaphase spread from the transgenic bull. Late-replicating DNA of synchronized fibroblasts cells was labeled by BrdU. Metaphase spreads were prepared on slides, fixed with methanol/acetic acid (3:1) and then used for fluorescent in situ hybridization (FISH) using the biotin-labeled probe corresponding to the plasmid containing the whole 8 kb-long Hsp70.1Luciferase transgene. After DNA counterstaining with propidium iodide in alkaline conditions, BrdU-rich bands appear as dark chromosomal bands while early replicated R bands fluoresce red. In these conditions the transgenic locus was detected on the Xp22 band by immunolabeling of the biotin probe and immunodetection with FITC-conjugated secondary antibodies. No other signal was detected on complete metaphase spreads. (B) (B') Partial metaphase spread of a female BSF731 fibroblast cell. Metaphases were prepared as above before (B) immunodetection or (B') DNA counterstaining. (B) After DNA denaturation and FISH with the DIG-labeled transgene probe, late incorporation of BrdU was immunolabeled with anti-BrdU antibodies and immunodetected with FITC-conjugated secondary antibodies; the DNA probe was immunolabeled with anti-DIG antibodies and detected with TRITC-conjugated secondary antibodies; in the case shown, the transgene was localized on the late-replicating X chromosome. Arrows indicate the position of the transgene.

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