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Knockout isoforem metalothioneinu pomocí metody CRISPR-Cas9 u adherentních tkáňových kultur
Duda, Jakub
It is not too long ago, that genome modifications were very complex, or, in some cases, almost impossible to achieve, but with the discovery and description of the CRISPR system in prokaryotes, a lot of breakthroughs came to the human knowledge, and thanks to some of these breakthroughs the CRISPR system has been modified to be used as a genome editing tool in eukaryotic organisms. Genetic modifications could in theory be the key to cancer therapy in modern pharmacy, and this thesis focuses on Metalmetallothionein (MT) proteins, which are commonly found in organisms and are beneficial to the organism (as inhibotors of oxidative stress, or as antioxidants binding heavy metals, for example), but can also be dangerous. Because, according to the isoform, and also the type of tissue, MTs can cause the development of cancer tissue and tumour growth, sometimes because of their presence, in other cases because of their absence. This thesis was focusing on the possible knock-out of all isoforms of MT at once, using CRISPR/Cas9 method, and thereby infucencing one of the very important factors in carcinogenesis. The result of this thesis is the achievement of knock-out of the MT3 isoform.
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Neuronal cell culture in vitro
Kohoutová, Šárka ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Neuronal cell cultures are in vitro cultures of dissociated neurons that have become an essential part of many neurobiological experiments in the last century. Cultured neurons not only allow to answer questions about their physiology under complex in vivo conditions, but also can serve as a model of neurodegenerative diseases. Neuronal cells can either be isolated directly from the nervous tissue of animals at the prenatal or adult stage of development, or they can be obtained through targeted manipulations of stem cells and secondary cell lines that lead to their neuronal differentiation. Primary neurons are considered the gold standard of neurobiological research not only because of their long tradition of cultivation, but also because primary neurons retain typical neuronal properties under in vivo conditions. There are several disadvantages associated with primary neurons, including the fact that fully differentiated neurons do not proliferate and are relatively demanding in terms of culture conditions For this reason, their role is often replaced by mitotically active secondary cell lines or stem cells. This bachelor thesis summarizes the knowledge about cell cultures used to study the functions of neuronal cells and highlights the advantages and disadvantages of their use. Key words Primary...
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Insertion of CRISPR system to carrot to enable genome editing
Chaloupský, Pavel
Main goal of this study was to develop carrot plants and callus lines with stable expression of Cas9 and Cpf1 genes. To achieve the thesis objective, transformation of Escherichia coli and Agrobacterium tumefaciens strains with different codon-optimized CRISPR constructs was carried out. Agrobacterium-mediated carrot callus transformation with CRISPR constructs, induction of somatic embryogenesis, and molecular confirmation of transgenic events in carrot were also part of the experiment executed within this thesis. By use of PCR-based genotyping, transgene was confirmed in 346 calli. Relative expression of Cas9 and Cpf1 in 15 selected lines was measured.
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The struggle for human dignity in the era of modern eugenics. Molecular genome editing tool CRISPR/CAS9 and its use in human genome therapy from the perspective of theological ethics
Auxt, Miroslav ; Štica, Petr (advisor) ; Fošum, Jan (referee)
The recent breakthrough discovery of the molecular genome editing tool CRISPR/CAS9 represents a complete revolution in the field of molecular biology, biomedicine and other related fields. It is a highly effective biomolecular tool, derived from the bacterial immune system, with which it is possible to introduce precise changes in the genomes of all organisms. The thesis is limited to the ethical evaluation of the use of CRISPR/CAS9 exclusively in human gene therapy. Thanks to its efficiency, simplicity, accuracy and low financial costs, the CRISPR/CAS9 editing tool, in compliance with ethical parameters, already has a broad spectrum of use in therapeutic procedures on somatic or body cells in the treatment of human genetically determined diseases without introducing a change into the future offspring of the given individual. In addition to great therapeutic potential, the application of CRISPR/CAS9 raises many ethical questions related to the possibilities of its further use, possibly misuse. Ethically problematic genetic procedures include: human hereditary genome editing, i.e. the targeted alteration of the genome of sex cells, progenitor cells and cells of early embryonic development stages with the therapeutic goal of eliminating a genetically determined disease associated with the...
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Analysis of Gretchen Hagen 3 gene family in tobacco BY-2 cell culture
Helusová, Lenka ; Müller, Karel (advisor) ; Pěnčík, Aleš (referee)
Auxin conjugation is one of the crucial metabolic processes regulating auxin activity in plant cells. Gretchen Hagen 3 (GH3) is a family of acyl amido synthetases that conjugates auxin with amino acids and belongs amongst important enzymes involved in auxin conjugation. Due to the existence of more sensitive methods to detect auxin metabolites and the current study of abiotic stress effects, research on GH3 enzymes is intensified these days. These enzymes are best known in thale cress (Arabidopsis thaliana), soya bean (Glycine max), rice (Oryza sativa). These models don't allow to study their activities in a biochemical way. Therefore, the aim of this work was to monitor the auxin metabolism in the established model tobacco BY-2 cell lines (Nicotiana tabacum). The NtGH3.1 and NtGH3.6 genes, which were shown to have a variability in their expression regulation by auxin, were targeted and mutated using tne CRISPR/Cas9 method. Mutations in the derived lines were detected by sequencing. In the derived lines, auxin metabolic profililing was analysed by LC/MS. Metabolic profiling showed a correlation between the NtGH3.6d form and the specific production of the metabolite oxIAA- Gln (N-(2-onindole-3-acetyl)-glutamine). The study of an eventual substitution of individual GH3 gene forms in mutant lines...
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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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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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