@misc{Liedtke2019, type = {Master Thesis}, author = {Liedtke, Victoria}, title = {Elucidating the role of DFS70/LEDGF/p75 in DNA damage response using CRISPR/Cas9 technology}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:co1-opus4-55913}, school = {BTU Cottbus - Senftenberg}, year = {2019}, abstract = {Lens epithelium derived growth factor (LEDGF), is known to be overexpressed in different solid cancers and cancer cell lines but is also involved in acquired immunodeficiency syndrome (AIDS) and diverse inflammatory diseases. Due to its chromatin-binding ability, it acts as a transcriptional coactivator, promoting anti-apoptotic pathways leading to increased tumor aggressiveness and chemotherapy resistance. In this thesis, the role of LEDGF in laryngeal cancer cells was investigated. Thus, a knock-out of LEDGF in HEp-2 cells was successfully generated by CRISPR/Cas9 technology. Moreover, LEDGF-deficient HEp-2 cells exhibited a decreased proliferation and migration, as well as an increased chemotherapy sensitivity towards topoisomerase II inhibitor etoposide. The DNA damage response was in more detail investigated, where LEDGF depleted cells showed a drastic reduction in the recruitment of downstream damage-response related proteins, more precisely replication protein A subunit of 32kDa (RPA32), and an increased amount of DNA fragmentation, highlighting the role of LEDGF in homology-directed DNA repair. In addition, a efficient method was established to allow genomic tagging of LEDGF at its C-terminus with EGFP which enables the monitoring of LEDGF in living cells. Therefore, an efficient Cas9-mediated DNA DSB induction was supplemented with a DNA template containing homology arms to the C-terminus as well as the EGFP sequence to allow homology-directed repair. Subsequently, EGFP-expressing cells were FACS sorted and single cells expanded. Almost all test clones showed a homozygous genotype for LEDGF-EGFP fusion which exhibited the typical nuclear pattern of LEDGF.}, subject = {LEDGF; Wachstumsfaktor; Linsenepithel; Kehlkopfkrebs; CRISPR/Cas-Methode; CRISPR/Cas9; Etoposide; DNA-Schadensantwort; FACS; CRISPR/Cas9; LEDGF; Etoposide; DNA damage response; FACS}, language = {en} }