Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2021
Erindi/veggspjald / Talk/poster E29
Höfundar / Authors: Salvör Rafnsdóttir (1), Li Zhang (2), Kimberley Anderson (1), Hans Tómas Björnsson (1,2,3).
Starfsvettvangur / Affiliations: 1. Laboratory of Translational Medicine, Faculty of Medicine, University of Iceland, 2. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, 3. Department of Genetics and Molecular Medicine, Landspítali University Hospital.
Kynnir / Presenter: Salvör Rafnsdóttir
Introduction: Targeted temperature management (TTM) is a neuroprotective treatment. Although, the treatment is widely used little is known about the mechanism behind its neuroprotective effect. Cold exposure causes increased transcription of cold shock genes. We hypothesize that TTM/cooling activates a specific neuroprotective pathway that is currently not fully elucidated. Materials and Methods: We have designed fluorescent indicators for 3 cold shock genes (CIRP, SP1 and RBM3). The indicator’s fluorescence is an indicator of their corresponding gene transcription at a given time. Cold exposure causes the fluorescence of the indicator to increase. We have done a genome wide CRISPR-Cas9 screen on a clonal HEK293 cell line with a stable expression of SP1 indicator. After the completion of the screen, we used FACS to isolate the top 5% fluorescent cells and bottom 5% fluorescent cells. Next the isolated samples and control samples were sequenced (NovaSeq) to compare sgRNA representation in sorted compared to unsorted. Results: After the genome wide CRISPR-Cas9 screen then we could see a shift of the fluorescence curve: 1) median fluorescence of cells shifted towards less fluorescence and 2) an increase in cells of extreme fluorescence. Conclusion: Our data indicate that the cold stress pathway has additional members that both inhibit (increased fluorescence) and activate (decreased fluorescence) the pathway. We are currently validating potential hits from this screen.