Generation of mice with targeted mutation of the mitochondrial DNA – Mito-Mouse

2 Meganucleases have been generated by Cellectis, targetting a specific sequence of mouse mtDNA. Their expression in mitochondria should select among the hundreds copies of mtDNA, those that present a mutation confering resistance to the action of Meganucleases, the other copies being elimlinated.

To date, we have generated molecular constructs allowing the targetted expression of the meganucleases into the mitochondria, associated or not to the GFP. We are now analysing their localisation into mitochondria and their nuclease activity on the mtDNA.

If we obtain the proof of concept that the Meganucleases specifically digest the mouse mtDNA, we will select cell and animal lines that are resistant to the effect of the Meganucleases in order to identify the mutations that provided the protection to the digestion by the Meganucleases.

None.
Collaboration contract with Cellectis issued by INSERM-Transfert

Manipulating the genome of organism is of crucial importance for studying development, physiological mechanisms, inherited diseases and genetics. To date, among mammals, only a single species, Mus musculus, allows targeted modifications of its nuclear genome by recombination of the chromosomal DNA. But all eukaryotes include a second genome located in mitochondria, with maternal mode of inheritance, which is responsible for inherited diseases and susceptibility to major pathologies as cancer, diabetes and Alzheimer and Parkinson diseases. Unfortunately, because of its high copy number per cell, manipulating the mtDNA has been facing a systematic limit, preventing the generation of animal models with mtDNA mutation reproducing human ones.

Here we propose to develop a technique to generate “à façon” cellular strains with mutated mtDNA. The concept involves the expression in mitochondria of Meganuclease restriction enzymes, which have been designed to cut a specific target sequence in the mtDNA. The Meganuclease should counter-select wild-type copies of this genome and allow selecting and amplifying rare mtDNA copies, mutated at the restriction site recognized by the enzyme. From the clones collected by this approach, the technique required to generate the corresponding trans-mitochondrial animal with the mutated mtDNA has been broadly described in the literature.

We have designed and obtained two meganucleases allowing to digest the mouse mtDNA at positions corresponding to mutated nucleotides responsible in human of the Leigh Syndrome and Leber Hereditary Optic Neuropathy. Our proposal aims to generate the two mito-mouse models for these diseases.
If effective, our experimental development will allow generating – and downstream studying - animal models with mutated mtDNA, without restriction of species, a technique that could also apply to the genome of plant organelles.