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Molecular Cytogenetics

, 9:28

Nuclear and chromosome architecture

Abstract

BackgroundThe centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells.

ResultsA panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation cenFISH, centromere protein immunofluorescence CENP-IF and multicolour FISH mFISH, and showed the usefulness of CENP-IF together with cenFISH followed by mFISH CENP-IF-cenFISH-mFISH with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric.

ConclusionsWe propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay DCA, prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.

KeywordsCentromere CENP-A CENP-C Immunofluorescence Fluorescence in situ hybridisation FISH Multicolour FISH mFISH Neocentromere Dicentric Dicentric chromosome assay DCA Human erythroleukaemia HEL cell line AbbreviationscenFISHcentromere fluorescence in situ hybridisation

CENP-IFcentromere protein immunofluorescence

Imm-FISHimmunofluorescence followed by FISH

mFISHmulticolour FISH

RTroom temperature

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Autor: Thian T. Beh - Ruth N. MacKinnon - Paul Kalitsis

Fuente: https://link.springer.com/







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