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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Měsíčková, Klára ; Fohlerová, Zdenka (referee) ; Svoboda, Ondřej (advisor)
The isolation of plasmid DNA is an important and often used method in microbiology. The isolation itself is preceded by preparation of bacterial competent cells and by amplification of the plasmids. In this stage, plasmids CHR2, ASAP1, ASAP-3, ASAP-5 and Kir2.1. are first amplified in E.Coli bacteria of the DH5 strain and then isolated through the method of phenol-chloroform extraction. Gel electrophoresis and transfection to cellular line HEK293 are used for determining the correctness of the isolation.
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Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell line
Jablonská, Dominika ; Čmiel, Vratislav (referee) ; Svoboda, Ondřej (advisor)
This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.
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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Bačovská, Kristýna ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
Plasmid DNA is commonly used in fields of molecular biology and genetic engineering. This work deals with methods of DNA isolation and topics related to this procedure. In the experimental part of the work, phenol-chloroform extraction is used. First of all, plasmids Channelrhodopsin-2, ASAP1 and Kir 2.1 were amplified in bacterial strain DH5. 22 isolations were accomplished and the yield was validated using gel electrophoresis and transfection to HEK293 cell line. The most successful isolation was the isolation of plasmid ASAP1, the overall percentage success rate was 30 %.
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Cardiac IKs channel: rate-dependence of the current magnitude
Kachan, Ksenia ; Čmiel, Vratislav (referee) ; Bébarová,, Markéta (advisor)
This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.
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Electrophysiological characterization of Kir2.1 membrane channel
Měsíčková, Klára ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.
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Plasmide DNA isolation from bacteria and transfection to HEK293 cell line
Skala, Kateřina ; Fohlerová, Zdenka (referee) ; Svoboda, Ondřej (advisor)
DNA isolation is one of the basic methods in molecular biology. There are several methods of DNA amplification and isolation. In this paper phenol-chloroform extraction of three plasmid types - Channelrhodopsin-2, ASAP1 and Kir 2.1 is used. Six plasmids were isolated in total. These plasmids are then validated using gel electrophoresis. Successfully isolated plasmids are then transfected to HEK293 cells and images taken on confocal microscope 24, 48 and 72 hours after transfection.
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Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell line
Sanetrníková, Dominika ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.
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Monitoring the success of transfection of cell line 293 HEK
Dvořák, Tomáš ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Diploma thesis is based on monitoring the succes of transfection of cell linie HEK293. In theoretical part are described principles of transfection methods, cell lines, vectors and reporter genes. HEK293 cells EBNA1 were used for practical part. It was studied the difference between GFP and EGFP plasmids. As well as using various transfection reagents under different culture conditions.
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Magnetic Nanoparticles for Targeted Delivery System of Plasmid into HEK293 cells
Bílek, Ondřej ; Svoboda, Ondřej (referee) ; Fohlerová, Zdenka (advisor)
This bachelor’s thesis deals with using of magnetic nanoparticles, SPIO nanoparticles and nanoparticles of commercial product MATra for transfection of ASAP plasmid into HEK293 cells and subsequent optimization of the process. The experimental part is enclosed with theorethical part that summarizes findings from the fields of synthesis of magnetic nanoparticles and their biomedical applications, theory dealing with possibilities of the insertion of foreign DNA or plasmids into the cell and of its efficiency evaluation.
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