Development of a transgenic animal model for measurement of intra-cellular ATP

by Friederike Kristin Wilbert

Institution: Freie Universitt Berlin
Year: 2017
Posted: 02/01/2018
Record ID: 2152376
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000105801


Mitochondriopathies are a group of clinically heterogeneous, mostly hereditary multisystemic disorders based on a deficient mitochondrial supply of ATP. Tissues with high energy demand, such as the brain or muscles, are predominantly affected. Symptoms include epileptic seizures, ataxia or muscle weakness. The underlying pathomechanisms are still not completely understood. In this context, the measurement of compartment-specific ATP levels represents an interesting approach. My doctoral thesis deals with the question whether the detection and quantification of intra-mitochondrial ATP in situ by means of the luciferase reaction would be possible. To explore this, I designed a gene construct containing the luciferase gene plus a mitochondrial targeting sequence and the antigenic fusion peptide FLAG-tag. Additionally, I added the Shine-Dalgarno sequence, necessary for translation in prokaryotes, and the Kozak consensus sequence including the eukaryotic start codon. The gene sequence of interest was inserted into the plasmid pENTR1A_DS. This vector is part of the Gateway Recombination System, a cloning tool based on the site-specific recombination pathway of the bacteriophage lambda being independent of appropriate restriction enzyme sites. This considerably facilitates cloning of fragments into different vector systems. I generated a vector for mammalian cell lines, (pcDNA3.2/V5_cox8a-flag-luc) and a targeting vector designed to insert the construct into the ROSA26 locus of the mouse (pROSA26_cox8a-flag-luc). I verified that transfection of HEK293 cells with the pcDNA3.2/V5_cox8a-flag-luc vector resulted in protein translation of a protein of about 70 kDa. By means of immunofluorescence, I identified the localization of the protein as intra-mitochondrial. I was able to confirm the functionality of the luciferase protein in transfected HEK293 and COS1 cells by means of a luciferin-luciferase assay. A luminometer detects the light emission in relative light units. Increasing amounts of the substrate luciferin led to a rise in light intensity. The second vector (pROSA26_cox8a-flag-luc) was constructed for the generation of a knock-in animal model with constitutive intra-mitochondrial luciferase expression. The transgenic mouse model will serve in future studies to bring forward the understanding of the pathomechanisms of mitochondriopathies. Mitochondriopathien sind eine Gruppe klinisch heterogener, meist genetisch bedingter Multi-System-Erkrankungen, die auf einer eingeschrnkten mitochondrialen Adenosintriphosphat (ATP)-Bereitstellung beruhen. Gewebe mit hohem Energiebedarf wie das Gehirn oder die Skelettmuskulatur sind vorrangig betroffen, Symptome wie epileptische Anflle, Ataxie und Muskelschwche sind hufig. Die zu Grunde liegenden Pathomechanismen sind bei weitem nicht vollstndig verstanden. In diesem Kontext wre die Messung kompartiment-spezifischer ATP-Spiegel ein interessanter Ansatz. Die vorliegende Dissertation behandelt die Fragestellung, ob die Erfassung und Quantifizierung des intramitochondrialen