Nanaka Torigoe, Qingyi Lin, Bin Liu, Yuichiro Nakayama, Aya Nakai, Megumi Nagahara, Fuminori Tanihara, Maki Hirata and Takeshige Otoi : Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.60, No.2, 2025.
(Summary)
Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.
(Keyword)
Animals / Cumulus Cells / Electroporation / Gene Editing / Female / Oocytes / CRISPR-Cas Systems / Fertilization in Vitro / Embryonic Development / Swine / Zygote / RNA, Guide, CRISPR-Cas Systems / Blastocyst / Galactosyltransferases / Embryo Culture Techniques
Qingyi Lin, Nanaka Torigoe, Bin Liu, Yuichiro Nakayama, Aya Nakai, Zhao Namula, Megumi Nagahara, Fuminori Tanihara, Maki Hirata and Takeshige Otoi : Efficient gene editing of pig embryos by combining electroporation and lipofection., Veterinary world, Vol.17, No.11, 2701-2707, 2024.
(Summary)
Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing.
Koki Takebayashi, Manita Wittayarat, Maki Hirata, Qingyi Lin, Zhao Namula, Nanaka Torigoe, Bin Liu, Megumi Nagahara, Aya Nakai, Takeshige Otoi and Fuminori Tanihara : Optimization of embryonic stage for aggregation to generate chimeric pigs using gene-edited blastomeres., In Vitro Cellular & Developmental Biology. Animal, 2024.
Aya Nakai, Akihito Tanaka, Hitoshi Yoshihara, Koji Murai, Takahito Watanabe and Katsuyuki Miyawaki : Blue LED light promotes indican accumulation and flowering in indigo plant, Polygonum tinctorium, Industrial Crops and Products, Vol.155, 112774, 2020.
(Keyword)
Polygonum tinctorium / Plant factory / Blue LED / Photoperiod / Flowering / Indican