Mitochondrial Transgenesis in mice Dr. Ziad Jaradat

Several human genetic disorders are caused by mitochondrial mutations and to model such disease it is necessary to introduce DNA into the mitochondrial genome. This CAN NOT be done by the regular DNA microinjection instead it is done as following; Donor cell lines carrying mitochondrial mutations are enucleated and then fused with recipient mouse zygotes.

The result is a transmitochondrial mice carry the mutation in every cell and passes the mutation through the female germline as the mitochondria in the sperm rarely contribute to the embryo. Procedure; A cell line must be produced carrying mitochondria with the appropriate mutation. This can be achieved by fusion between cells depleted in mitrochondrial DNA (mtDNA) and synaptosomes from aged mice which often contained mutated mtDNA Resulting cell lines are called cybrids as their cytoplasm is a hybrid of two cell lines Resulting cell lines are called cybrids as their cytoplasm is a hybrid of two cell lines

Remove the nucleus from cybrid cells so they do not introduce any nuclei into the egg. This can be done by centrifugation in the presence of cytochalasin B. Wash the enucleated cybrids Collect zygotes from donor females. With a micromanipulator drill a hole through the zona pellucida of the zygote and place the cybrids in the perivitelline space. Let embryos stay in culture for a brief recovery.

Embryos containing cybrids are washed in medium that promotes fusion and placed between electrodes of a fusion chamber. A long AC pulse (225 V, s) is used to align the cytoplastis and oocytes, then a brief DC pulse (2500 V, 20 ms) is used to induce fusion which occurs within the next hour. Fused embryos are implanted into pseudopregnant foster mothers and raised to term.

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