Methods for the detection of genome instability derived from replication stress in primary mouse embryonic fibroblasts.

Replication stress, with its subsequent genome instability, is a hallmark of cancer from its earliest stages of development. Here, we describe assays that are sufficiently sensitive to detect intrinsic replicative stress and its consequences in primary mouse embryonic fibroblasts. First, we explain the non-denatured DNA fiber assay, a powerful tool to directly measure DNA replication kinetics via the dual-labeling of active replication forks. Then, we describe the cytokinesis-block micronucleus assay, which can be combined with detection of 53BP1 nuclear bodies to measure the levels of replication-associated genome instability carried over into G1 phase of the cell cycle.