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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Zadražil, Zdeněk (referee)
CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent B-lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, mainly due to complications from CLL, but most patients live 5-10 years. However, with disease progression significantly increases level of miR-155, which is known as oncomiR. MicroRNAs (miRNAs) represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets (similarly as other miRNAs). We hypothesized that by gene editing of CLL cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Methods: We used CRISPR/Cas9 method for gene editing, which enables the deletion of mature miR-155 sequence in the genome of leukemic B-cells. CRISPR/Cas9 plasmid was transferred to the leukemic B-cell cell line HG-3 via nucleofection. Clones with successful transfer of...
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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Zadražil, Zdeněk (referee)
CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent B-lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, mainly due to complications from CLL, but most patients live 5-10 years. However, with disease progression significantly increases level of miR-155, which is known as oncomiR. MicroRNAs (miRNAs) represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets (similarly as other miRNAs). We hypothesized that by gene editing of CLL cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Methods: We used CRISPR/Cas9 method for gene editing, which enables the deletion of mature miR-155 sequence in the genome of leukemic B-cells. CRISPR/Cas9 plasmid was transferred to the leukemic B-cell cell line HG-3 via nucleofection. Clones with successful transfer of...
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Selected aspects of redox metabolism in leukemogenesis
Myšáková, Michaela ; Pimková, Kristýna (advisor) ; Kuželová, Kateřina (referee)
Blood cancers are caused by the accumulation of mutations in haematopoietic stem cells. This creates a malignant clone that has a selection advantage due to improved survival and unrestricted proliferation, a process of leukaemia development called leukemogenesis. Leukemogenesis is a complex process and it is difficult to identify a single mutation that is responsible for the transformation of haematopoietic cells. In addition to transcriptional deregulation caused by oncogenic fusion proteins, mutations in specific genes that regulate critical signaling pathways play a critical role in leukemogenesis. Examples of such genes include mutations in the isocitrate dehydrogenase 1 and 2 genes (mutIDH1/2). These genes are thought to play an important role in the development of leukaemia, as indicated by their increasing frequency in the progression of myelodysplastic syndrome to acute myeloid leukaemia. The functions of mutIDH1/2 include epigenetic regulation, changes in metabolism and redox homeostasis. It has been shown that regulation of reactive oxygen species (ROS) production and elimination, so-called redox homeostasis, is important for the proper function of haematopoietic stem cells and its disruption is a frequent phenomenon accompanying malignant transformation of these cells. Some mutations,...
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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Mráz, Marek (referee)
Markéta Sypecká CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years. However, every stage of this disease has significantly higher level of miR-155, which is known as oncomiR. Micro RNAs represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets. We hypothesized that by gene editing of CLL B - cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Method we use for gene editing is CRISPR/Cas9, which enables to delete sequence of mature miR-155 in genome of leukemic B-cells. Methods: CRISPR/Cas9, nucleofection, qRT-PCR, FACS Results:We achieved to isolate clone that bears one allelic deletion (miR-155-/+) in sequence for mature...
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