Bivariate flow cytometry of farm animal chromosomes: a potential tool for gene mapping

Dixon, S. C.; Miller, Nigel; Carter, N. P.; Tucker, Elizabeth M.

doi:10.1111/j.1365-2052.1992.tb00132.x

Cited by 25 publications

(5 citation statements)

References 9 publications

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“…A solution to this problem has come from the use of the ZOO-FISH procedure in a bidirectional way developed first using pig chromosomes [Goureau et al, 1996]. Authors were able to flow-sort porcine chromosomes, which mostly differ in size and morphology, and to prepare chromosome-specific paints for each porcine chromosome [Dixon et al, 1992;Yerle et al, 1993]. The hybridization of human chromosome-specific paints on porcine metaphases completed by the reverse experiment, e.g.…”

Section: Zoo-fish For Identification Of Conserved Chromosomal Blocks mentioning

confidence: 99%

Fluorescence in situ Hybridization Applied to Domestic Animal Cytogenetics

Rubeš

Pinton

Bonnet-Garnier

et al. 2009

Cytogenet Genome Res

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The aim of this article is not to present an exhaustive review of molecular cytogenetics applications in domestic animal species, but more to illustrate the considerable contribution of these approaches in diagnostics and research in economically important species. A short presentation of the main applications of molecular cytogenetics in humans points out the domains in which it has become an essential tool and underlines the specificities attached to this species in comparison to farm animals. This article is devoted to outlining the current resources available in domestic species and to some examples of fluorescence in situ hybridization applications in the cattle, pig, horse and avian species. From a clinical point of view, these examples illustrate the advantages of FISH for the study of chromosomal abnormalities (identification, characterization and estimation of their effects). Other applications of molecular cytogenetics are also illustrated, particularly ZOO-FISH, an approach which allows the determination of chromosome homologies between species. Finally, a specific emphasis was placed on the usefulness of molecular cytogenetics for the analysis of species such as poultry, which harbour a complex karyotype.

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Section: Zoo-fish For Identification Of Conserved Chromosomal Blocks mentioning

confidence: 99%

Fluorescence in situ Hybridization Applied to Domestic Animal Cytogenetics

Rubeš

Pinton

Bonnet-Garnier

et al. 2009

Cytogenet Genome Res

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“…The ovine chromosomes flow sorting was early reported in 1992, and only the first three large metacentric chromosomes and five other clusters could be resolved 30 . A high-resolution flow karyotype for sheep was obtained in 1997, from which nearly all chromosomes have been isolated and identified 19 .…”

Section: Discussionmentioning

confidence: 99%

Highly efficient synchronization of sheep skin fibroblasts at G2/M phase and isolation of sheep Y chromosomes by flow cytometric sorting

Yao

Zhang

Liu

et al. 2020

Sci Rep

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that with whole genome sequencing, enrichment of Y chromosomes before sequencing might simplify data analysis and reduce sequencing costs. Flow cytometric sorting is attractive because of its capacity to purify single chromosomes in large numbers. Moreover, in recent years, sequencing technology has advanced to permit the sequencing of single chromosomes of interest isolated by flow cytometric sorting. This method has been successfully applied in humans 5,6 , gorillas 1 , Chinese hamsters 7 , and some plant species 8-11. To date, this application has not been reported with domestic animals. The Y chromosomes of many domestic animals, including the sheep Y chromosome, have not been fully assembled and characterized. In this study, we isolated and enriched sheep Y chromosomes by flow cytometric sorting and performed NGS of sorted Y chromosomes, which laid a foundation for the sequencing, assembly, and analysis of the sheep Y chromosome. Chromosomes with different sizes and AT/GC contents can be separated by flow cytometric sorting after staining with two specific fluorescent dyes (Hoechst 33258-AT specific binding and chromomycin A3-GC specific binding) 12,13. However, chromosomes with small differences in size and GC content are not easily segregated. The so-called bivariate flow karyotype is generated based on the fluorescence intensity of the two dyes, and the purity of flow-sorted chromosomes can be greater than 95% with high-quality samples and favorable conditions 12,14. Further, the flow cytometric sorting of chromosomes has been applied to DNA hybridization, DNA libraries, physical mapping, and chromosome sequencing 12. The quality of chromosome samples is critical in chromosome flow cytometric sorting 15,16. A high-quality chromosome sample contains a large number of single chromosomes in a suitable chromosome isolation buffer. For mammals, chromosome samples are usually prepared from metaphase cells. To obtain a large number of metaphase cells in a short time, cells need to be synchronized in the G2/M phase of the cell cycle. Although the preparation of chromosome suspensions from mammalian cell lines 17,18 , primary cells 19,20 , and peripheral blood lymphocytes 21,22 has been reported, conditions for cell-cycle synchronization differ among species, and this is especially the case when primary cells differ. Just like most of mammalian species, the sequences of the ovine-Y chromosome are not available. In this study, we established a highly efficient synchronization method for G2/M phase of sheep fibroblasts suitable for sheep chromosomes flow sorting. We successfully isolated and enriched ovine Y chromosomes by flow cytometric sorting and performed NGS. The purity of sorted sheep Y chromosome was analyzed using NGS data for the first time. This study demonstrates the potential application of flow cytometry for genome and chromosome research and lays a foundation for future sequencing and annotation of the ovine Y chromosome. Results Establishment, purification, and culture of sheep primary skin fibroblas...

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“…Generally, the paints can be prepared by flow sorting multiple copies of specific chromosomes, which is followed by degenerate oligonucleotide-primed PCR (DOP-PCR) amplification. Cattle chromosomes, however, show poor separation in bivariate flow cytometry [ 56 ]. Therefore, laser microbeam microdissection and laser pressure catapulting procedures completed with DOP-PCR were preferred by Kubickova et al [ 57 ] and lately by Frolich et al [ 58 ] for the construction of chromosome-specific painting probes in cattle.…”

Section: Chromosomal Aberrationsmentioning

confidence: 99%

Chromosomal Aberrations in Cattle

Holečková

Schwarzbacherová

Galdíková

et al. 2021

Genes

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Chromosomal aberrations and their mechanisms have been studied for many years in livestock. In cattle, chromosomal abnormalities are often associated with serious reproduction-related problems, such as infertility of carriers and early mortality of embryos. In the present work, we review the mechanisms and consequences of the most important bovine chromosomal aberrations: Robertsonian translocations and reciprocal translocations. We also discuss the application of bovine cell cultures in genotoxicity studies.

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Bivariate flow cytometry of farm animal chromosomes: a potential tool for gene mapping

Cited by 25 publications

References 9 publications

Fluorescence in situ Hybridization Applied to Domestic Animal Cytogenetics

Fluorescence in situ Hybridization Applied to Domestic Animal Cytogenetics

Highly efficient synchronization of sheep skin fibroblasts at G2/M phase and isolation of sheep Y chromosomes by flow cytometric sorting

Chromosomal Aberrations in Cattle

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