In this article, we describe detailed protocols for the isolation and transfer of spindle–chromosomal complexes between mature, metaphase II-arrested oocytes. In brief, the spindle–chromosomal complex is visualized using a polarized microscope and extracted into a membrane-enclosed karyoplast. Chromosomes are then reintroduced into an enucleated recipient egg (cytoplast), derived from another female, by karyoplast–cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA (mtDNA), from another female. This approach yields developmentally competent oocytes suitable for fertilization and producing embryonic stem cells or healthy offspring. The protocol was initially developed for monkey oocytes but can also be used in other species, including mouse and human oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Chromosome transfer between the cohorts of oocytes isolated from two females can be completed within 2 h.
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This work was supported by start-up funds from the Oregon National Primate Research Center, the Oregon Stem Cell Center and grants from the National Institutes of Health HD057121, HD059946, HD063276, HD047721, HD047675, RR0000163 and U54 HD18185.
The authors declare no competing financial interests.
Micromanipulation procedures for chromosome transfer involve: 1) MII oocyte positioning for chromosome-spindle detection, laser assisted zona drilling and chromosome-spindle aspiration into a karyoplast; 2) karyoplast exposure to HVJ-E; 3) and karyoplast transfer into a perivitelline space of a cytoplast. (MOV 6059 kb)
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Tachibana, M., Sparman, M. & Mitalipov, S. Chromosome transfer in mature oocytes. Nat Protoc 5, 1138–1147 (2010). https://doi.org/10.1038/nprot.2010.75
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