Mitochondrial Genetics And Animal Modeling

Mammalian mitochondria contain between one and approximately ten copies of a closed, circular, super-coiled, double-stranded DNA that is bound to the inner mitochondrial membrane and is not associated with histones or a scaffolding protein matrix. The mtDNAs of all vertebrates are highly conserved and quite small 16.5 kb in length) in comparison to the nuclear genome. Mammalian mitochondria have their own genetic systems, replete with a unique genetic code, genome structure, transcriptional and translational apparatus, and tRNAs. Perhaps, because of a postulated less-extensive mitochondrial DNA (mtDNA) repair system and because of the absence of protective histones, the mitochondrial genome is subject to an increased sensitivity to mutations due to metabolic (e.g., oxidative stress) and environmental (e.g., toxins, mutagens, and UV light) sources. Mitochon-drial genes encode for 13 of the protein subunits that function in the mitochondrial oxidative phosphorylation system, along with two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). Accordingly, directed modification of mitochondrial genes and/or their function would provide a powerful tool in production agriculture.[1]

Cytoplasmic-based traits in domestic animals have included growth, reproduction, and lactation. In addition, mitochondrial restriction fragment-length polymorphisms (RFLPs) were identified and associated with specific lactational characteristics in a number of dairy cattle lineages. The matrilineal inheritance of mammalian mtDNA has also been used to advantage in studies exploring the timing and geography of domestication events, as recently demonstrated for horses, where multiple domestication events appear to have occurred in the Eurasian steppe.[2] In addition, metabolic and cellular abnormalities in humans were correlated to mutations arising exclusively within the mitochondrial genome. Indeed, various diseases have been associated with mtDNA point mutations, deletions, and duplications (e.g., diabetes mellitus, myocardiopathy, and retinitis pigmentosa) as well as age-associated changes in the functional integrity of mitochondria (as seen in Parkinson's, Alzheimer's, and Huntington's diseases). As such, for both agricultural and biomedical research efforts, the ability to manipulate the mitochondrial genome and to regulate the expression of mitochondrial genes would provide one possible mode of genetic manipulation and therapy.

The creation of heteroplasmic transmitochondrial animals has developed along three lines: 1) direct mito-chondrial injection into oocytes or embryos; 2) embryonic stem (ES) cell-based technologies; and 3) in relation to karyoplast or cytoplast transfer (including consequences associated with nuclear transfer or cloning experimentation; Table 1). These techniques have illustrated model systems that will provide a greater understanding of mito-chondrial dynamics, leading to the development of genetically engineered production animals, and therapeutic

Table 1 Methods for creating mitochondrial modifications in animals

Method

Heteroplasmy/ homoplasmy detected

Germline transmission

Limitations

Mitochondrial injection into ova Heteroplasmy

Karyoplast fusion Heteroplasmy (nuclear transfer)

Karyoplast or cytoplast Yes transfer into ES cells and transfer

Cytoplast/ooplasm transfer Heteroplasmy

Sperm mediated ?

Yes Low level heteroplasmy

Yes Varying efficiencies using

PEG or electrofusion Yes Availability of germline competent/efficient cell lines Yes Varying efficiencies and low level heteroplasmy ? Rare event, aberrant recombination, or programmed destruction postfertilization strategies for human metabolic diseases affected by aberrations in mitochondrial function.

As described in a number of recent reports,[3'4] nuclear-encoded genes and knock-out modeling have been informative in identifying novel models in mitochondrial disease pathogenesis as well as critical pathways associated with mitochondrial function. With initial characterization of these nuclear gene-encoded models, our search for a greater understanding of mitochondrial interactions and function would eventually lead us to a desire to develop methodology for mitochondria and mitochondrial gene transfer. As a first step, efficient methods to introduce foreign or altered mtDNA or genomes into somatic or germ cells would be needed.

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