Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Abstract

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

BLM helicase regulates DNA repair by counteracting RAD51 loading at DNA double-strand break sites

“…Telomere DNA FISH analysis was performed and analyzed as previously described ( Misenko and Bunting, 2014 ). B cells were activated for 24 h and treated with 250 nM mitomycin C (Sigma Aldrich), 2 µM olaparib (KU0059436; Selleckchem), or 4 µM cisplatin (Sigma Aldrich) for 16 h. Cells were arrested in metaphase with 100 ng/ml colcemid (Sigma Aldrich) for 1 h. Cells were collected, incubated in hypotonic solution (0.075 M KCl) for 15 min at 37°C, and fixed in a 3:1 methanol/acetic acid solution (three washes) and stored overnight at −20°C.…”

53BP1 ablation rescues genomic instability in mice expressing ‘RING‐less’ BRCA1

“…Resting B lymphocytes were isolated from mouse spleens and cultured with LPS (25 μg/ml, Sigma) and IL‐4 (5 ng/ml, Sigma) as described . After 36‐h growth, the cells were mock‐treated or treated with olaparib (2 μM) overnight, then arrested with 10 μg/ml colcemid for 1 h. Metaphase fixation and telomere PNA‐FISH was performed as previously described .…”

SIRT 7 promotes genome integrity and modulates non‐homologous end joining DNA repair

“…Sister chromatid exchange assays were performed as previously described (Misenko & Bunting, 2014) using primary B cells.…”