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The role of ETV6-RUNX1 fusion protein in the sensitivity of leukemic cells to L-asparaginase
Staněk, Petr ; Starková, Júlia (advisor) ; Burjanivová, Tatiana (referee)
Translocation t(12;21) with the presence of the fusion gene ETV6-RUNX1 (TEL-AML1) is the most common chromosomal aberration found in acute lymphoblastic leukemia in childhood. The occurrence of the ETV6-RUNX1 is associated with excellent prognosis and high sensitivity to the treatment with the enzyme L-asparaginase (ASNase). Resistance to the drug aggravates the outlook of the patient and increases the risk of treatment failure, therefore, the CLIP working group has been for a long time involved in the identification of the mechanism of action of ASNase and the origin of the resistance to it. This thesis follows previous findings of the group and is devoted to the analysis of the importance of ETV6-RUNX1 and signalization and metabolic changes accompanying shifts in the L-asparaginase resistance. In the first part of the thesis, the knockout clones with stable increased resistance to ASNase have been established thanks to the CRISPR/Cas9 system, which created frameshift in the fusion gene. The accomplishment in this regard and removal of the fusion protein was confirmed on the level of DNA, mRNA a protein expression. The presence of other significant chromosomal aberrations affection the sensitivity to ASNase was ruled out by the means of SNP analysis. In the second part of the project, the signalization...
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Applications of CRISPR-Cas9 for genetic engineering in parasitic protists
Ročeň, Milan ; Tachezy, Jan (advisor) ; Rada, Petr (referee)
The CRISPR / Cas system serves as a cellular defense mechanism that protects bacteria and archaea from foreign DNA, especially bacteriophages. Its product forms a ribonucleoprotein complex, whose components are sgRNA and Cas endonucleases. Using sgRNAs, which are complementary to foreign DNA, this complex recognizes the DNA and Cas endonucleases induce double-strand breaks. This method is applied both in primary research, where it is applicable for functional analysis of proteins and the study of gene expression, as well as in applied research, where it can apply, for example, in the production of genetically modified organisms or genetically attenuated vaccines. This work summarizes the current knowledge of the CRISPR / Cas system and its application for gene manipulation in parasitic protists.
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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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The role of tissue specific isoforms of subunit 4 in assembly and function of cytochrome c oxidase
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Stibůrek, Lukáš (referee)
Oxidative phosphorylation apparatus (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process, occurring in the inner mitochondrial membrane, is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. Cox4 subunit, participating in OXPHOS regulation, is an early-assembly state subunit, which is necessary for incorporation of Cox2 catalytic subunit, thus for assembly of catalytically functional COX enzyme. Moreover, regulated expression of two isoforms (Cox4i1, Cox4i2) of Cox4 subunit is hypothesized to optimize respiratory chain function according to tissue oxygen supply. However, the functional impact of the isoform switch for mammalian tissues and cells is still only partly understood. In the present thesis, unique HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) was prepared employing novel CRISPR CAS9-10A paired nickase technology and further characterized. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of majority of Cox subunits resulting in total absence of fully assembled COX. Moreover, detected Complex I subunits as well as the content of assembled Complex I were decreased in COX4i1/4i2 KO clones. On the...
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Preparation of nanoparticles for hepatitis B viral therapy
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Žáčková Suchanová, Jiřina (referee)
Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery
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Use of RNAi and CRISPR systems in genetic modifications of parasitic protists
Kaiserová, Veronika ; Votýpka, Jan (advisor) ; Stojanovová, Darja (referee)
In organisms, RNA interference serves as a defence mechanism against foreign nucleic acids. RNAi has a negative effect on translation, via the binding of small non-coding molecules to the complementary region of mRNA, resulting in its degradation. CRISPR, a new method of genetic engineering, is based upon modulating genetic expression via creating double-stranded breaks in target DNA, aided by a ribonucleoprotein complex, consisting of the prokaryotic endonuclease Cas9 and sgRNA. Both of the aforementioned methods can be utilised in functional analysis of proteins and the characterisation of metabolic pathways in organisms of interest. This work summarises the current state of knowledge regarding RNAi and CRISPR and their use in genome editing of parasitic protists.
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Generation of large animal models using genome editing
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Kašpárek, Petr (referee)
The principle of gene engineering is the intervention to the DNA of the studied organism. After the discovery of the programmed endonucleases, there has been a great expansion of this technique and it also accelerated the possibilities to create large animal models. Until recently, large animal models were very difficult to be generated. These endonucleases include zinc finger nuclease (ZFN), transcription activator like effector nuclease (TALEN) and CRISPR/Cas9. All endonucleases produce locally specific splicing in the targeted segment of the genome. This splicing is most easily corrected by the non-homologous ends joining (NHEJ), so then it is possible to create a so -called knock-out (KO) model. The second type of repair is homologous recombination (HR) using a DNA template with homologous arms. This makes it possible to create a knock-in (KI) model that cannot be created without specific endonucleases in large animal models due to the low natural HR. This work summarizes the history, technique and the use of programmed endonucleases for the creation of large animal models. These models have a great use in biomedicine, mostly in preclinical research, they are also significant in agriculture and even in the environment protection. Key words: large animal model, transgenesis, genome editing,...
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Visual system development in Platynereis dumerilii: insight from genetic engineering approach
Dobiášovská, Ivana ; Kozmik, Zbyněk (advisor) ; Vopálenský, Václav (referee)
Gene regulatory networks, underlying the molecular regulation of eye development are conserved across many animal phyla. Genes from the Pax family of transcription factors are one of the most conserved members through the evolution, regulating the development of crucial parts of eye, including the photoreceptor cells. Pax transcription factors are considered to be regulators of opsins, molecules providing the conversion of the light stimulus into the electrochemical signalisation in the photoreceptors cells. In this thesis, pax6 and pax2/5/8 transcription factors are investigated as potential regulators of eye development in Platynereis dumerilii. pax6 and pax2/5/8 transcription factors are tested as potential regulators of the r-opsin in Platynereis, based on the observed early expression onsets of these genes. Wild-type expression analysis of pax6 and pax2/5/8 using the whole mount RNA in-situ hybridization is provided, accompanied by the initial analysis of the Platynereis pax6 knockout line. pax6 heterozygote mutants are shown to be viable and able to reproduce, however, homozygote mutation of pax6 in Platynereis is lethal. Our data suggest that transcription factors pax2/5/8, otx and six3 are not regulated by the pax6 in Platynereis. Concerning the r-opsin present in the Platynereis eyes, pax6...
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Generation and analysis of double deficient transgenic mice for kallikrein-related peptidase 5 and kallikrein-related peptidase 14
Hanečková, Radmila ; Sedláček, Radislav (advisor) ; Fulková, Helena (referee)
Kallikrein-related peptidases (KLKs) constitute a highly conserved serine protease family. Based on in vitro experiments, KLKs are predicted to play an important role in a number of physiolog- ical and pathophysiological processes. However, their role in vivo remains not fully understood, partially due to a lack of suitable animal models. In this work, we aim to prepare a KLK5 and KLK14 double-deficient mouse model. Both KLK5 and KLK14 were proposed to be involved in epidermal proteolytic networks critical for maintaining skin homeostasis. However, both KLK5 and KLK14 single-deficient mouse models show minimal or no phenotype, likely due to similar substrate specificity resulting in functional compensation. Double-deficient mice cannot be easily obtained by crossing due to localization of the Klk5 and Klk14 genes within the same locus on chromosome 7. We report that KLK5 and KLK14 double-deficient mice were success- fully generated, mediated by transcription activator-like effector nucleases (TALENs) targeting Klk14 by microinjection of TALEN mRNA into KLK5-deficient zygotes. Furthermore, we show that KLK5 and KLK14 double-deficient mice are viable and fertile. We believe that these novel mouse models may serve as a useful experimental tool to study KLK5 and KLK14 in vivo.
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