Mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) are a significant source of morbidity and mortality, yet only supportive care is available to affected patients. This lack of effective therapies can be partly attributed to the lack of faithful animal models for mitochondrial diseases caused by mutations in mtDNA. Two critical obstacles to the availability of mouse models of these diseases are 1) the lack of mouse mtDNA mutations homologous to those found in human disease, and 2) the lack of a method to efficiently generate such mutations. Therefore, the long-term GOALS of the proposed studies are 1) to overcome these obstacles in order to enable routine modeling of human diseases caused by mtDNA mutations, and 2) to provide mouse models of mitochondrial disease to the research community. In this application, we propose to leverage expertise developed at the University of South Alabama (mutagenesis of mouse mtDNA) and the Children?s Hospital of Philadelphia (transmitochondrial mouse models) towards generating models of human diseases caused by mutations in mitochondrially-encoded tRNA genes. Using the tools and techniques developed in the previous funding cycle and in preliminary studies, we will 1) generate an arrayed library of mouse clones carrying mutations in mtDNA, 2) use it to perform a targeted screening for mutations in tRNALeuUUR and in tRNALys, and 3) use these mutations as well as tRNA mutations isolated by us previously for the generation and characterization of transmitochondrial animals. It is anticipated that, if successful, the proposed studies will deliver a powerful impetus to mitochondrial research in general and to research on mitochondrial disease in particular by providing the mitochondrial research community with (1) mouse model(s) for mitochondrial disease caused by mutations in tRNA genes, (2) an arrayed library of mouse clones suitable for targeted screening for mtDNA mutations of interest as well as tools and protocols for generating and screening such libraries, and (3) cell line(s) carrying mouse homolog(s) of human pathogenic mtDNA mutations. In this competitive revision, we request funds to procure equipment that will dramatically facilitate the generation and storage of an arrayed library of mouse cell lines containing mutations in mtDNA. While the generation of a collection of mutant mammalian cell lines is not without precedent, the generation of an arrayed library of cell lines with mutations in mtDNA has not been attempted before. In the previous funding cycle, our attempts to generate and screen a large representative library of mouse mtDNA mutants were hampered by low throughput of cryopreservation and sequencing protocols. The equipment that will be procured with the requested funds will introduce semi-automation into our attempts at cryopreservation of the clones, and will enable storage of clones in an 8x12 array format, which is amenable to medium- and high- throughput manipulation and screening.
In this competitive revision of our R01 OD010944 application (funded on 01/01/2016), we request funds to purchase equipment for the arraying and liquid nitrogen storage of clones with mutations in mtDNA, which are the subject of the parent grant. This revision broadens the scope of the original award and requests support for ongoing research activities to facilitate the generation of animal models of mitochondrial disease. Purchased equipment will be installed in the Gene Delivery Core and therefore will be accessible (and beneficial) to all researchers who need to generate and store a large number of mammalian cell line clones (e.g., CRISPR- Cas9 generated libraries of ES cell or iPS cell mutants).
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