小鼠四倍体器官的制备

Tetraploidy occurs infrequently in mammals but remains widespread in amphibians. Blastocyst complementation using xenogeneic transplantation of tetraploid embryonic stem cells (4N-ESCs) represents a promising approach to mitigate organ shortages, yet robust generation of fully reconstituted organs in mammalian hosts remains elusive. In this study, CRISPR/Cas9, the Cre-LoxP system, and blastocyst complementation were combined to generate tetraploid mouse liver, heart, and pancreatic tissues. 4N-ESCs (tdTomato-labeled) were established and shown to maintain stable pluripotency and tetraploidy, as confirmed by karyotyping and immunofluorescence analyses. Subsequently, these cells were microinjected into Hhex- and Pdx1-deficient blastocysts and Nkx2.5 lineage-ablated blastocysts, which were engineered to lack relevant organ-forming lineages. Tetraploid pups exhibited significantly reduced body mass and organ mass (liver and heart) relative to diploid controls (P