Functional decoding of signaling dynamics in single immune cells

National Institute On Aging

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Abstract

We acquire real time dynamics data from a sensitive laser scanning microscope system with a built-in long-term tissue incubation capability. The NF-kappaB subunits RelA and c-Rel are transcriptional regulators that mediate the responses of cells to stimulation of various receptors by different pathogenic stimuli. We investigated how the patterns of RelA and c-Rel signaling dynamics differ in multiple cell types including tissue-resident macrophages and bone marrow derived macrophages. Despite the lockdown and restrictions imposed in the COVID-19 pandemic, our projects progressed slowly albeit with delays and setbacks. We have developed four novel knock-in mouse strains since 2016. Each strain expresses a fluorescent NF-kappaB subunit fusion from the native genomic locus, by CRISPR-Cas genome engineering. We screened pups for correct expression and integration of the targeting constructs. We have also performed extensive crossbreeding to generate homozygous colonies of double knockin mice where both RelA and c-Rel genes are endogenously labeled with a fluorescent protein. Simultaneous live cell imaging of the two NF-B subunits in the same cells will reveal unprecedented insight about how the distinct dimers of NF-B interact with each other to achieve selective gene expression programs in immunity and cell differentiation.