62 Triploid complementation: a potential alternative approach to its tetraploid counterpart

Tetraploid (4N) complementation is an effective approach to generate genetically engineered mice from embryonic stem cells (ESCs) in one generation. However, its application has been restricted by its low efficiency and has been rarely achieved in other species. Conventional methods, such as blastocyst injection and morula aggregation, achieved survival rates of 4.3%–6% in mice (Wang et al. 1997 Mechanisms of Development 62.2, 137–145). Although laser-assisted 8-cell embryo injection has improved the survival rate to 8%–40% (Poueymirou et al. 2007 Nat. Biotechnol. 25, 91–99), these rates were highly dependent on the strains of both the donors and recipients. Triploid (3N) conceptuses exhibited developmental characteristics similar to their 4N counterparts in that they failed to develop into fetuses but successfully formed fetal membranes (Niemierko et al. 1981 Development 66.1, 81–89). This outcome suggests that 3N embryos could also serve as recipients for ESC injection. In this study, we investigated 3N complementation, aiming to enhance the efficiency of ESC-derived mice and potentially extend it to other mammals. We used cytochalasin B to generate 3N mouse embryos by IVF. An average of 57.19 ± 14.19% (n = 5) fertilized embryos displayed three pronuclei after treatment. Among these, 58.33% of embryos maintained the 3N karyotype by the blastocyst stage, while the rest reverted to 2N. We then aggregated denuded presumptive 3N morula with mouse ESCs expressing GFP under the constitutive and ubiquitous CMV promotor. A remarkable 81.81% of the aggregated embryos showed GFP+ cell clumps at the position of the inner cell mass. Immunofluorescence assay confirmed that the GFP+ cells overlapped with OCT4 signals, indicating the successful contribution of ESCs to the inner cell mass. Aggregated embryos cultured to Day 10.5 (Day 0 = fertilization) were composed primarily of GFP+ cells, which were localized exclusively in the epiblasts of the egg cylinder and absent in the trophoblastic cap and mural trophectoderm. In conclusion, our findings demonstrate that mouse ESCs effectively integrate with 3N embryos and predominantly contribute to the embryo proper. This method has the potential to generate 3N-complemented embryos with high contribution from ESCs, offering a promising alternative for producing ESC-derived mice in the F0 generation.