Dermal fibroblasts from a proband female donor were cultured in 4-well dishes under standard conditions until they reach confluency. Confluent cells were synchronized in the G0/G1 phase of the cell cycle by culture in medium with low serum (DMEM/F12 medium with 0.5% FBS) for 2–4 days before SCNT. Enucleations, cell fusion, and artificial activations were performed. Briefly, meiotic metaphase II (MII) spindles were visualized under polarized microscopy and removed. Next, a disaggregated fibroblast was aspirated into a micropipette, exposed briefly to HVJ-E extract (Cosmo Bio LTD #ISK-CF-001-EX) and placed into the enucleated oocyte perivitelline space. After cell fusion, the SCNT oocytes were subjected to artificial activation.
Liang D., Mikhalchenko A., Ma H., Marti Gutierrez N., Chen T., Lee Y., Park S.W., Tippner-Hedges R., Koski A., Darby H., Li Y., Van Dyken C., Zhao H., Wu K., Zhang J., Hou Z., So S., Han J., Park J., Kim C.J., Zong K., Gong J., Yuan Y., Gu Y., Shen Y., Olson S.B., Yang H., Battaglia D., O’Leary T., Krieg S.A., Lee D.M., Wu D.H., Duell P.B., Kaul S., Kim J.S., Heitner S.B., Kang E., Chen Z.J., Amato P, & Mitalipov S. (2023). Limitations of gene editing assessments in human preimplantation embryos. Nature Communications, 14, 1219.
Other organizations :
Oregon Health & Science University, Shandong University, CHA University, Institute for Basic Science, Asan Medical Center, University of Ulsan, Ulsan College, BGI Group (China), Shanghai Institutes for Biological Sciences, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Renji Hospital, Shanghai Jiao Tong University
Culture medium for fibroblast synchronization (DMEM/F12 medium with 0.5% FBS)
Duration of fibroblast synchronization (2-4 days)
Enucleation of oocytes
Fusion of fibroblast with enucleated oocyte
Artificial activation of SCNT oocytes
dependent variables
Completion of fibroblast synchronization in G0/G1 phase
Successful removal of meiotic metaphase II (MII) spindle from oocytes
Successful fusion of fibroblast with enucleated oocyte
Successful artificial activation of SCNT oocytes
control variables
Standard culture conditions for fibroblast cell culture
Visualization of meiotic metaphase II (MII) spindle using polarized microscopy
positive controls
HVJ-E extract (Cosmo Bio LTD #ISK-CF-001-EX) for cell fusion
negative controls
Not mentioned
Annotations
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