Erschienen:
[Erscheinungsort nicht ermittelbar]: eScholarship, University of California, 2012
Sprache:
Englisch
Entstehung:
Hochschulschrift:
Dissertation, eScholarship, University of California, 2012
Anmerkungen:
Beschreibung:
Mitochondrial DNA (mtDNA) with deleterious mutations can cause severe inherited diseases due to defective energy metabolism. Studies have shown that mutated mitochondrial genomes alone are sufficient to cause diseased phenotypes in cells with wild-type nuclear genomes. This suggests that the transfer of mitochondria containing wild-type mtDNA into mutant cells may offer a way to rescue defective cell respiration. Several previous attempts to rescue diseased phenotypes using cell fusion have been either unsuccessful or irreproducible. Our development of a device called the photothermal nanoblade provides a new approach to rescue mtDNA defects. In this technique, a non-focused laser pulse is used to excite a titanium coating at the tip of a glass micropipette positioned adjacent to the membrane of a single cell. The laser pulse creates an explosive vapor bubble in the surrounding aqueous cell medium that collapses in less than 1µs and which generates a shear force to cut an adjacent plasma cell membrane with high precision. Cargo, such as wild-type mitochondria, are then delivered through the hollow bore of the titanium tipped micropipette into the cell cytoplasm. This technique is, therefore, distinct from standard microinjection and can deliver large objects, such as mitochondria and intracellular bacterial pathogens, into recipient cells with minimal damage. The goal of this project is to improve the respiration defect of mtDNA-depleted osteosarcoma rho 0 cells by photothermal nanoblade delivery of wild-type mitochondria, and to validate by studying the resulting recovery in oxidative phosphorylation. We have isolated respiring yet uncoupled wild-type mitochondria from an engineered DsRedMito HEK293 line, and shown successful delivery into rho 0 cells with high efficiency, demonstrating the feasibility of photothermal nanoblade as a large cargo delivery tool. Data collected from multiple isolation-delivery experiments followed by media selection did not select a population with improved rho 0 respiration. A series of mitochondria characterization ruled out the possibility of mitochondrial autophagy, suggesting that chemiosmotic coupled isolated mitochondria could be the key to achieve cellular uptake of the delivered mitochondria.