Resurrection of chromosomes from frozen animals by single chromosome transfer into mouse oocytes

Reviving extinct animals offers a crucial opportunity to recover lost or unknown genetic resources, yet cloning methods are unsuitable because they depend on intact donor nuclei and abundant oocytes or recipients from closely related species. To overcome these constraints, we explored a chromosome level revival strategy. Blood cells from rat carcasses stored at - 30 °C for over one year were introduced into enucleated mouse oocytes, where the rat nuclei underwent premature chromosome condensation. Microtubule polymerization inhibition enabled dispersion of rat chromosomes within the ooplasm, allowing isolation of individual chromosomes by micromanipulation. Each chromosome was subsequently transferred into an intact mouse oocyte, followed by intracytoplasmic sperm injection using GFP-transgenic mouse sperm. Embryos were cultured to the blastocyst stage, yielding 17 ES cell lines, two of which carried 41 chromosomes. Spectral karyotyping confirmed the presence of rat chromosome 9 alongside a full set of normal mouse chromosomes. These ES cells generated chimeric mice exhibiting GFP based chimerism across multiple organs. Histological analyses further demonstrated expression of numerous genes located on rat chromosome 9 within chimera mouse. This study demonstrated that a single chromosome from a frozen extinct species can be functionally revived and its transcriptional activity assessed within an interspecies oocyte.