A Modified Protocol for Accurate Detection of Cell Fusion-Mediated Premature Chromosome Condensation in Human Peripheral Blood Lymphocytes

Abstract

Summary When interphase cells are fused with mitotic cells, loosely distributing chromatins in nuclei are induced to form prematurely condensed chromosomes. In this paper we report a modified protocol to unequivocally detect prematurely condensed chromosomes in human peripheral blood lymphocytes that were fused with mitotic Chinese hamster ovary (CHO) cells. To examine cell fusion-mediated premature chromosome condensation (PCC), we conducted morphological analysis by differential interference contrast microscopy and molecular cytogenetic analysis by fluorescence in situ hybridization using pan-centromeric and telomeric peptide nucleic acid (PNA) probes. These modified procedures may serve to improve the usefulness of the technique of PCC in cytogenetic investigations.

Detection and Automated Scoring of Dicentric Chromosomes in Nonstimulated Lymphocyte Prematurely Condensed Chromosomes After Telomere and Centromere Staining

“…The introduction of peptide nucleic acid (PNA) probes to the scoring of radiation-induced CAs, with short hybridization times, high specificity, increased signal intensity, and lower cost, could open new horizons for this application (9)(10)(11). However, the long, brightly staining, interstitial telomere sequences of CHO cells impede the detection of the much shorter and more dimly stained telomere sequences in human lymphocytes (12).…”

Assessing the Applicability of FISH-based Prematurely Condensed Dicentric Chromosome Assay in Triage Biodosimetry

“…The prematurely condensed chromosomes tended to have a fuzzy appearance after the DNA denaturing procedure, due probably to the loosely packed chromatin architecture (Suto et al 2013a). In the present study, a combination of modified cytogenetic techniques was devised to optimize the analysis of prematurely condensed chromosomes.…”

“…Cell fusion-mediated PCC was conducted following the previously described protocol (Suto et al 2013a). Two kinds of experimental procedures were designed for repair kinetic and dose-effect analyses in G0 mononuclear cells irradiated with gamma rays.…”

Chromatin Tagging Systems Contribute to Live Imaging Analyses for Chromatin Dynamics

“…Fluorescent in situ hybridization (FISH) has revealed chromosomal loci or regions of DNA sequences on mitotic chromosomes (Suto et al 2012, Ikeda et al 2013, Kuroki et al 2013, Ruan et al 2013, Yokomi et al 2013, Suto et al 2013, Kantek et al 2015, Shibata and Hizume 2015. Three dimensional (3D) FISH can visualize the location of DNA sequences in interphase nuclei and show the alteration of chromatin structure (Matsunaga 2016).…”