Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

Line-scanning temporal focusing microscopy (LineTFM) is capable of imaging biological samples more than 10 times faster than two-photon laser point-scanning microscopy (TPLSM), while achieving nearly the same lateral and axial spatial resolution. However, the image contrast taken by LineTFM is lower than that by TPLSM because LineTFM is severely influenced by biological tissue scattering. To reject the scattered photons, we implemented LineTFM using both structured illumination and uniform illumination combined with the HiLo post-processing algorithm, called HiLL microscopy (HiLo-Line-scanning temporal focusing microscopy). HiLL microscopy significantly reduces tissue scattering and improves image contrast. We demonstrate HiLL microscopy with in vivo brain imaging. This approach could potentially find applications in monitoring fast dynamic events and in mapping high resolution structures over a large volume.

Nucleosomes package genomic DNA into chromatin. By regulating DNA access for transcription, replication, DNA repair, and epigenetic modification, chromatin forms the nexus of most nuclear processes. In addition, dynamic organization of the chromatin fiber underlies both regulation of gene expression and evolution of chromosomes into individualized sister objects which can segregate cleanly to different daughter cells at anaphase. This collaborative review shines a spotlight on technologies that will be crucial to interrogate key questions in chromatin and chromosome biology including state-of-the-art microscopy techniques, tools to physically manipulate chromatin, single-cell methods to measure chromatin accessibility, computational imaging with neural networks and analytical tools to interpret chromatin structure and dynamics. In addition, this review provides perspectives on how these tools can be applied to specific research fields such as genome stability and developmental biology, and to test concepts such as phase separation of chromatin. eTOC blurbIn this collaborative review, Agbleke et al., discuss the development and application of new technologies to probe chromatin and chromosome biology questions. The authors examine new chromatin concepts, drawing on perspectives from researchers within the chromatin community as well as from those in adjacent fields.