GFP/AID tagging of endogenous BLM for rapid protein depletion
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To study how mitotic functions of BLM helicase contribute to genome stability, we genereated chromosomally stable HCT116 cell lines, in which BLM protein can be rapidly degraded in a cell-cycle specific manner. Using the CRISPR/Cas9 technology, a homology donor encoding an auxin-inducible degron (AID), GFP and a selection marker was inserted at the last coding exon of the endogenous BLM gene. To generate the homology donor, a modular cloning system was set up using the Golden Gate assembly strategy. One homozygous BLM-AID clone as well as two heterozygous BLM-AID clones were identified and successful tagging was confirmed by redcued BLM signal upon auxin induction in these cells.
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Created: 7th Feb 2022 at 16:44
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Our Research Unit addresses an important question on the origin of chromosome instability (CIN), which causes structural as well as numerical chromosome aberrations. Importantly, CIN and increased levels of chromosome aberrations are closely associated with many human diseases including cancer, neurodegenerative diseases and age-related syndromes and can act as key drivers for disease development and progression. An important condition that causes structural chromosome instability (S-CIN) leading ...
Projects: SP-3: A statistical modeling approach to identify common triggers for replication stress and mitotic chromosome missegregation, SP-1: Deciphering signaling pathways that promote accurate chromosome segregation after replication stress, SP-2: Molecular mechanisms of replication stress-induced mitotic chromosome missegregation, SP-4: Centrosome integrity as a determinant of replication stress and mitotic dysfunction, SP-5: Impaired chromosome integrity caused by mutations in members of the BTR complex, SP-6: Proteomics of mitotic inter sister chromatid junctions, SP-8: The causes of replication stress in response to whole chromosomal aneuploidy, SP-Z: NGS-based approaches for systematic analysis of genomic and chromosome instability Bernd Wollnik, Göttingen, SP9: Mutual impact of replication origin firing regulation and mitotic chromosome segregation
Web page: https://for2800.de/
Replication stress is a key driver of genomic instability giving rise to human disease, for instance cancer or developmental disorders. It is caused by a variety of defects such as lesions blocking replication (e.g. DNA interstrand crosslinks) or processes that lead to the exhaustion of DNA precursors. This generates under-replicated DNA and replication intermediates that can be rescued by repair processes requiring homologous recombination between sister chromatids. Often, cells exposed to ...
Programme: FOR2800
Public web page: https://for2800.de/cases/sub-project-6-proteomics-of-mitotic-inter-sister-chromatid-junctions/
Genomic instability is a hallmark of cancer and can be frequently induced by replication stress. Incomplete repair of replication intermediates can lead to persistent linkages between sister chromatides, detectable during mitosis as ultra-fine anaphase bridges (UFBs). The active resolution of UFBs via several UFB factors are crucial to prevent mitotic catastrophe, chromosomal missegregations or aneuploidy. Previous studies have already revealed that UFBs are processed by a distinct set of proteins, ...
Submitter: Angela Wieland
Studies: GFP/AID tagging of endogenous BLM for rapid protein depletion, Sister chromatid exchange assay of BLM depletable cell lines
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