主催: Society for Reproduction and Development
Although Somatic cell nuclear transfer (SCNT) is the preferred approach for producing transgenic large animals, there are still numerous technical challenges, which substantially limit widespread application. The objective of the present study was to systematically investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene NT dairy goats, thereby providing insights for improving transgenic cloning efficiency. In our study, 3-month-old goat fibroblasts were transfected with hLF-gene mammary gland expression vector. Positive colonies were identified, expanded, and cryopreserved. After cell cycle synchronization treatment, cell cycle and apoptosis were analyzed by flow cytometry. Meanwhile, nuclear transfer-derived kids were identified by PCR identification. In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 d of serum starvation or 2 d of contact inhibition; kidding rate of recipients in serum starvation group was higher than that in contact inhibition group (18 vs 0%). Oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-month-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.
