Production of second-generation sheep clones via somatic cell nuclear transfer using amniotic cells as nuclear donors.

Somatic Cell Nuclear Transfer (SCNT) has transformed animal genetic improvement, gene-editing in model production, xenotransplantation, and conservation efforts for endangered species. However, SCNT-derived embryos occasionally display developmental abnormalities, and following embryo transfer, the miscarriage rate is high. Gene-edited fetuses may experience birth defects, resulting in decreased survival rates. Correct selection of nuclear donor cells is essential for the success of somatic cell cloning. Fibroblasts are the most commonly used cells, but their rapid proliferation increases the risk of genetic mutation, impairing embryo development and production. Conversely, amniotic cells have slower proliferation rates, decreasing the mutation risk during cultivation. Amniotic cells are thus better SCNT candidates than fibroblasts because they offer genomic stability, low tumorigenic and teratogenic risks, reduced immunogenicity, high differentiation potential, ease of accessibility, and fewer ethical concerns. Cells derived from first-generation gene-edited animals exhibit stable genetic structures, reduced susceptibility to genetic alterations and artificial modifications, closely resembling natural cells, and enhanced compatibility with SCNT procedures. Amniotic cells derived from gene-edited sheep fetuses used as nuclear donor cells for SCNT successfully recloned three healthy second-generation gene-edited sheep. Using amniotic cells as nuclear donor cells for SCNT did not significantly alter embryo cleavage rates, blastocyst formation, or fetal birth compared to edited fibroblasts (p > 0.05). However, fetal survival rates were significantly higher than edited fibroblasts (p < 0.05). The results support the potential of amniotic cells as SCNT alternatives, suggesting a promising strategy to improve gene-edited fetus survival rates using first-generation gene-edited sheep-derived amniotic cells.