Production of Cloned Calves Following Nuclear Transfer with Cultured Adult Mural Granulosa Cells1

TLDR

The study aims to advance nuclear transfer technology by incorporating targeted genetic modifications for biomedical or agricultural applications. The authors performed adult somatic cell nuclear transfer using cultured mural granulosa cells, fusing their nuclei to enucleated metaphase‑II cytoplasts 4–6 h before activation (FBA) and, in some cases, recloning morulae by fusing blastomeres to S‑phase cytoplasts. FBA produced a higher blastocyst development rate (27.5 % vs 13 %) and, after transfer of 100 blastocysts, yielded ten surviving heifer calves (10 % term survival), whereas recloned embryos had lower survival and none reached term; DNA confirmed genetic identity to the donor cow.

Abstract

Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4–6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1–2 blastocysts on Day 7 with FBA (27.5 ± 2.5%) than with recloning (13.0 ± 3.6%; p < 0.05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest.