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Chromosome orientation fluorescence in situ hybridization to study sister chromatid segregation in vivo

Abstract

Previously, assays for sister chromatid segregation patterns relied on incorporation of 5-bromo-2′-deoxyuridine (BrdU) and indirect methods to infer segregation patterns after two cell divisions. In this study, we describe a method to differentially label sister chromatids of mouse cells and to directly assay sister chromatid segregation patterns after one cell division in vitro and in vivo by adaptation of the well-established CO-FISH technique. BrdU is incorporated into newly formed DNA strands, which are then subjected to photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially labeled sister chromatids in postmitotic cells are visualized using fluorescence microscopy, and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires 4 d for in vivo mouse tissues and 2 d for in vitro–cultured cells.

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Figure 1: Uniform orientation of mouse chromosomes as shown by four-color CO-FISH.
Figure 2: Time-course experiments to ensure that BrdU incorporation is for one complete cell cycle.
Figure 3: Hourly injections of BrdU are necessary to ensure single-stranded chromosome targets for strand-specific hybridization.

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Acknowledgements

We thank S. Poon for helping proofread the paper. The authors also thank S. Selig, T. de Lange and R.C. Wang for help with CO-FISH protocols. The original study was funded in part by grants from the Canadian Institutes of Health Research (RMF-92093), the Michael Smith Foundation for Health Research, the Canadian Cancer Society Research Institute and the Terry Fox Foundation. We are grateful to the British Columbia Cancer Agency, the Canada Foundation for Innovation, the British Columbia Knowledge Development Fund, the British Columbia Cancer Foundation, the Blusson Fund of the University of British Columbia and the Mahon family for funding the infrastructure that enabled this work.

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E.F. wrote the paper and created the figures; E.C. performed most of the CO-FISH procedure and image acquisition for the original study and proofread the paper; A.H. performed the animal experiments in the original study and proofread the paper; P.M.L. originally developed this procedure, verified the protocol and helped write this paper.

Corresponding author

Correspondence to Peter M Lansdorp.

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The authors declare no competing financial interests.

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Falconer, E., Chavez, E., Henderson, A. et al. Chromosome orientation fluorescence in situ hybridization to study sister chromatid segregation in vivo. Nat Protoc 5, 1362–1377 (2010). https://doi.org/10.1038/nprot.2010.102

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