Mouse models of human genetic diseases have been critical for confirming that particular mutations cause disease, for obtaining a detailed molecular understanding of the pathophysiology of these diseases, and for developing and testing potential therapies. Technical limitations, however, currently prevent us from generating accurate mouse models of human diseases caused by mutations in our mitochondria! DNA. Although several groups have shown that they can generate mice that contain mitochondrial genomes isolated from mouse tissue culture cells, the technology has not yet been developed that allows us to introduce specific nucleotide changes into mouse mitochondrial genomes. We are proposing to overcome this technical limitation. We have cloned the complete mouse mitochondrial genome stably in E. coli and have shown that we can introduce essentially any desired mutation into these clones. We have also shown that we can introduce these engineered mitochondrial genomes into the mitochondria of yeast cells devoid of their own mtDNA and that these mouse mtDNA genomes are replicated accurately in these cells. We now propose to use these transgenomic yeast mitochondria as vectors for transferring the engineered mtDNA genomes back into the mitochondrial networks of mouse tissue culture cells, which will then be used to generate mice with modified mitochondrial genomes.
Our specific aims for this project are to 1) develop and optimize methods for using transgenomic yeast mitochondria as mitochondrial delivery vectors that will efficiently fuse to mouse mitochondrial networks, and to 2) determine how to best screen or select for mouse cells that have been transformed with modified mitochondrial genomes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21NS052612-01
Application #
6962761
Study Section
Special Emphasis Panel (ZRG1-GTIE (01))
Program Officer
Tagle, Danilo A
Project Start
2005-07-15
Project End
2007-04-30
Budget Start
2005-07-15
Budget End
2006-04-30
Support Year
1
Fiscal Year
2005
Total Cost
$165,785
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Pathology
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Yoon, Young Geol; Koob, Michael D (2012) Nonreplicating intracellular bacterial vector for conjugative DNA transfer into mitochondria. Pharm Res 29:1040-5
Yoon, Young Geol; Koob, Michael Duane (2011) Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker. J Genet Genomics 38:173-9
Yoon, Young Geol; Koob, Michael D; Yoo, Young Hyun (2011) Mitochondrial genome-maintaining activity of mouse mitochondrial transcription factor A and its transcript isoform in Saccharomyces cerevisiae. Gene 484:52-60
Yoon, Young Geol; Yang, Yi-Wei; Koob, Michael D (2009) PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in Escherichia coli. Biotechnol Lett 31:1671-6
Yoon, Young Geol; Koob, Michael D (2008) Selection by drug resistance proteins located in the mitochondria of mammalian cells. Mitochondrion 8:345-51
Yoon, Young Geol; Haug, Christopher L; Koob, Michael D (2007) Interspecies mitochondrial fusion between mouse and human mitochondria is rapid and efficient. Mitochondrion 7:223-9
Yoon, Young Geol; Koob, Michael D (2005) Transformation of isolated mammalian mitochondria by bacterial conjugation. Nucleic Acids Res 33:e139