Article
Biochemistry & Molecular Biology
Fuminori Tanihara, Maki Hirata, Zhao Namula, Manita Wittayarat, Lanh Thi Kim Do, Qingyi Lin, Koki Takebayashi, Hiromasa Hara, Megumi Nagahara, Takeshige Otoi
Summary: This study successfully generated biallelic GHR-mutant small-stature pigs using CRISPR/Cas9 system. The backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain, which can greatly contribute to biomedical research.
MOLECULAR BIOLOGY REPORTS
(2023)
Article
Biochemistry & Molecular Biology
Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Osamu Sawamoto, Takeshi Kikuchi, Takeshige Otoi
Summary: The study successfully utilized a CRISPR/Cas9 system to simultaneously target GGTA1, CMAH, and B4GALNT2 in in vitro-fertilized zygotes, achieving the one-step generation of multiple gene-edited pigs without xenoantigens. Although the experiments resulted in the generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs, these gene-edited pigs were genetic mosaics, indicating further refinement is needed to completely knock out all xenoantigens.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Article
Reproductive Biology
Sergio Navarro-Serna, Martxel Dehesa-Etxebeste, Celia Pinerio-Silva, Raquel Romar, Jordana S. Lopes, Adolfo Lopez de Munain, Joaquin Gadea
Summary: This study successfully generated a pig model of limb girdle muscular dystrophy type R1 (LGMDR1) by optimizing porcine oocyte electroporation. Electroporation, as a faster and easier method compared to microinjection, showed comparable rates of embryo development and mutation parameters.
Article
Cell Biology
Mingyun Lee, Kwang-Hwan Choi, Jong-Nam Oh, Seung-Hun Kim, Dong-Kyung Lee, Gyung Cheol Choe, Jinsol Jeong, Chang-Kyu Lee
Summary: The study revealed that SOX2 plays a crucial role in inner cell mass formation and cell proliferation in early-stage porcine embryogenesis. Disruption of SOX2 inhibits the expression of inner cell mass-related genes and reduces total cell number; while overexpression of SOX2 greatly increases blastocyst cell number but decreases the inner cell mass/trophectoderm ratio.
CELL PROLIFERATION
(2021)
Article
Agriculture, Dairy & Animal Science
Celia Pineiro-Silva, Sergio Navarro-Serna, Ramses Belda-Perez, Joaquin Gadea
Summary: Lipofection treatment, which involves encapsulating the CRISPR/Cas9 system into vesicles and introducing them into cells, can be a simple and inexpensive method for producing genetically modified pigs. It has similar effects on embryo development and mutation rates as the electroporation technique when used in zona-pellucida-intact porcine oocytes. However, increasing the concentration of liposomes-CRISPR/Cas9 system complexes in the media has a detrimental effect on embryo development parameters, possibly indicating a toxic effect.
Article
Agriculture, Dairy & Animal Science
Koki Takebayashi, Manita Wittayarat, Qingyi Lin, Nanaka Torigoe, Bin Liu, Maki Hirata, Megumi Nagahara, Fuminori Tanihara, Takeshige Otoi
Summary: This study found that genetic mosaicism is a limitation of the electroporation method used for transferring CRISPR-Cas9/gRNA into porcine zygotes. The combination of fertilization with gene-deficient spermatozoa and gene editing using electroporation did not improve the efficiency of embryo genetic modification. These findings suggest that electroporation alone is sufficient for genome modification.
ANIMAL SCIENCE JOURNAL
(2023)
Article
Multidisciplinary Sciences
Beate Rieblinger, Hicham Sid, Denise Duda, Tarik Bozoglu, Romina Klinger, Antonina Schlickenrieder, Kamila Lengyel, Krzysztof Flisikowski, Tatiana Flisikowska, Nina Simm, Alessandro Grodziecki, Carolin Perleberg, Andrea Bahr, Lucie Carrier, Mayuko Kurome, Valeri Zakhartchenko, Barbara Kessler, Eckhard Wolf, Lutz Kettler, Harald Luksch, Ibrahim T. Hagag, Daniel Wise, Jim Kaufman, Benedikt B. Kaufer, Christian Kupatt, Angelika Schnieke, Benjamin Schusser
Summary: Research on genetically modified animals has mostly focused on mice, but also includes species like pigs that are more physiologically similar to humans, and cross-species comparisons with phylogenetically distant species like chickens. CRISPR-Cas9 is a versatile genetic editing method applicable across various species. The successful generation of transgenic chickens and pigs that express Cas9 in all organs has confirmed the functionality of Cas9 across different target genes, cell types, and in vivo applications.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Environmental Sciences
Kaidi Jin, Baolu Zhang, Qianqian Jin, Zhongqiang Cai, Lei Wei, Xiaomei Wang, Yanxin Zheng, Baoyu Huang, Meiwei Zhang, Yitao Qi, Yaqiong Liu, Xiaotong Wang
Summary: This study attempted to deliver the CRISPR/Cas9 system into embryos of C. angulate by electroporation, demonstrating the feasibility of this method for gene editing in oysters.
FRONTIERS IN MARINE SCIENCE
(2021)
Article
Agriculture, Dairy & Animal Science
Maki Hirata, Manita Wittayarat, Zhao Namula, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Chommanart Thongkittidilok, Fuminori Tanihara, Takeshige Otoi
Summary: Liposome-mediated gene transfer is a feasible method for establishing a gene targeting framework and producing mutant animals even in laboratories without specialized equipment. However, the efficiency and factors affecting the system in mammalian oocytes and embryos remain unclear.
Article
Biochemistry & Molecular Biology
Ahmed K. Mahdi, Juan F. Medrano, Pablo J. Ross
Summary: We investigated the possibility of single-step genome editing in small ruminants using CRISPR-Cas9 zygote electroporation. We found that electroporation was more efficient than microinjection and successfully disrupted target genes in sheep and goat embryos.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Agriculture, Dairy & Animal Science
Kamlesh Kumari Bajwa, Meeti Punetha, Dharmendra Kumar, Prem Singh Yadav, Chares R. Long, Naresh L. Selokar
Summary: In this study, the optimization of electroporation conditions allowed for successful editing of the ITGB2 gene in adult buffalo fibroblasts, achieving high editing efficiency. These electroporation conditions can be applied to various biotechnological research applications for buffalo genes.
ANIMAL BIOTECHNOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Chaya T. L. Yuen, Dawn G. L. Thean, Becky K. C. Chan, Peng Zhou, Cynthia C. S. Kwok, Hoi Yee Chu, Maggie S. H. Cheung, Bei Wang, Yee Man Chan, Silvia Y. L. Mak, Anskar Y. Leung, Gigi C. G. Choi, Zongli Zheng, Alan S. L. Wong
Summary: Researchers have enhanced editing accuracy and reduced off-target edits by re-engineering KKH-SaCas9 with a Y239H mutation and a set of additional mutations, demonstrating the feasibility of multi-domain combinatorial mutagenesis to optimize editing fidelity.
NUCLEIC ACIDS RESEARCH
(2022)
Article
Reproductive Biology
Gyeong-Min Gim, Kyeong-Hyun Uhm, Dong-Hyeok Kwon, Min-Ji Kim, Dae-Jin Jung, Dae-Hyun Kim, Jun-Koo Yi, Jae-Jung Ha, Soo-Young Yum, Woo-Jae Son, Ji-Hyun Lee, Ji-Hyun Park, Kil-Young Song, Won-Wu Lee, Goo Jang
Summary: This study assessed the germline transmission of MSTN mutations in F0 cattle and demonstrated successful transmission of the mutations to the next generation. Additionally, the study showed the possibility of further gene editing using electroporation in embryos fertilized with the mutant sperms.
Article
Agriculture, Multidisciplinary
Guang-Ming Xiang, Xiu-ling Zhang, Chang-jiang Xu, Zi-yao Fan, Kui Xu, Nan Wang, Yue Wang, Jing-jing Che, Song-song Xu, Yu-lian Mu, Kui Li, Zhi-guo Liu
Summary: A study showed that efficient transgene knock-in using the CRISPR/Cas9 system in the COL1A1 gene did not affect mRNA expression or global gene expression, and had no adverse effects on cell proliferation. This indicates that the COL1A1 locus can serve as a safe harbor for the insertion of foreign genes.
JOURNAL OF INTEGRATIVE AGRICULTURE
(2023)
Article
Agriculture, Dairy & Animal Science
Gyeong-Min Gim, Kyeong-Hyeon Eom, Dong-Hyeok Kwon, Dae-Jin Jung, Dae-Hyun Kim, Jun-Koo Yi, Jae-Jung Ha, Ji-Hyun Lee, Seong-Beom Lee, Woo-Jae Son, Soo-Young Yum, Won-Wu Lee, Goo Jang
Summary: Electroporation has been successfully used to edit genes and produce mutated cattle. This method eliminates the need for technically demanding methods such as microinjection and somatic cell nuclear transfer.
JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY
(2023)
Article
Agriculture, Dairy & Animal Science
Maki Hirata, Manita Wittayarat, Zhao Namula, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Chommanart Thongkittidilok, Fuminori Tanihara, Takeshige Otoi
Summary: Liposome-mediated gene transfer is a feasible method for establishing a gene targeting framework and producing mutant animals even in laboratories without specialized equipment. However, the efficiency and factors affecting the system in mammalian oocytes and embryos remain unclear.
Article
Agriculture, Dairy & Animal Science
Chommanart Thongkittidilok, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Thi Kim Lanh Do, Zhao Namula, Maki Hirata, Fuminori Tanihara, Takeshige Otoi
Summary: This study found that supplementation with three antioxidants (beta-ME + CGA + curcumin) during oocyte maturation culture can enhance blastocyst formation and improve blastocyst quality.
REPRODUCTION IN DOMESTIC ANIMALS
(2022)
Article
Agriculture, Dairy & Animal Science
Qingyi Lin, Quynh Anh Le, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Chommanart Thongkittidilok, Osamu Sawamoto, Takeshi Kikuchi, Takeshige Otoi
Summary: This study developed an efficient method for liquid storage of in vitro-derived porcine blastocysts at ambient temperature for 24 hr. The results showed that storing the blastocysts in a Cell-S solution at 25 degrees Celsius maintained their survival and development, and supplementation with curcumin improved their development rate.
REPRODUCTION IN DOMESTIC ANIMALS
(2022)
Article
Veterinary Sciences
Zhao Namula, Maki Hirata, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Naoaki Yoshimura, Fuminori Tanihara, Chommanart Thongkittidilok, Takeshige Otoi
Summary: The study showed that weakening the ZP does not affect the developmental competence, mutation rate, or mutation efficiency of electroporated zygotes, while ZP removal has a detrimental effect on embryonic development but may increase the mutation rate.
VETERINARY MEDICINE AND SCIENCE
(2022)
Article
Agriculture, Dairy & Animal Science
Zhao Namula, Quynh Anh Le, Manita Wittayarat, Qingyi Lin, Koki Takebayashi, Maki Hirata, Lanh Thi Kim Do, Fuminori Tanihara, Takeshige Otoi
Summary: This study aimed to evaluate the effects of a combination of microinjection (MI) and electroporation (EP) on the biallelic mutation rates for triple-gene knockout in porcine blastocysts. The results showed that the combination of MI and EP did not improve the mutation efficiency or biallelic mutation rates, but MI treatment before EP was better at reducing mortality in porcine zygotic gene.
Article
Agriculture, Dairy & Animal Science
Koki Takebayashi, Manita Wittayarat, Qingyi Lin, Maki Hirata, Naoaki Yoshimura, Nanaka Torigoe, Megumi Nagahara, Lanh Thi Kim Do, Fuminori Tanihara, Takeshige Otoi
Summary: CRISPR/Cas9 technology has greatly influenced the efficiency and effectiveness of genetic modifications. The study found that electroporation alone significantly increased the frequency of biallelic mutations, while lipofection alone required the removal of the zona pellucida to enable CRISPR/Cas9-based genome editing. Additionally, additional lipofectamine treatment did not improve the mutation rates.
REPRODUCTION IN DOMESTIC ANIMALS
(2022)
Article
Agriculture, Dairy & Animal Science
Quynh Anh Le, Manita Wittayarat, Zhao Namula, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Lanh Thi Kim Do, Takeshige Otoi
Summary: The study aimed to develop a technique combining microinjection, electroporation, and transfection methods to enhance the mutation efficiency of triple-gene knockout in porcine zygotes mediated by the CRISPR/Cas9 system.
Article
Reproductive Biology
Qingyi Lin, Mutsumi Aihara, Akihiro Shirai, Ami Tanaka, Koki Takebayashi, Naoaki Yoshimura, Nanaka Torigoe, Megumi Nagahara, Takeo Minamikawa, Takeshige Otoi
Summary: This study examined the effects of UV-C irradiation on embryo development and the growth of bacteria and fungus. It was found that 228 nm UV-C can effectively inhibit the growth of bacteria and fungus, with minimal impact on the development and quality of porcine embryos. However, 260 nm UV-C has a greater detrimental effect on embryo development.
Article
Cell Biology
Fuminori Tanihara, Maki Hirata, Zhao Namula, Lanh Thi Kim Do, Naoaki Yoshimura, Qingyi Lin, Koki Takebayashi, Tetsushi Sakuma, Takashi Yamamoto, Takeshige Otoi
Summary: By introducing a precise point mutation into the porcine insulin (INS) gene using the CRISPR/Cas9 system via electroporation, genetically modified pigs with the desired point mutation were successfully established as suitable islet donors for xenotransplantation.
FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY
(2023)
Article
Agriculture, Dairy & Animal Science
Qingyi Lin, Quynh Anh Le, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Osamu Sawamoto, Takeshi Kikuchi, Takeshige Otoi
Summary: This study aimed to develop a simple method for short-term preservation of in vitro-produced porcine zygotes at 25°C for up to 2 days. The results showed that the Cell-W solution was the most efficient for 24-hour storage of porcine zygotes at 25°C, with no apparent effects on blastocyst quality. However, as storage duration increased, blastocyst formation rates significantly decreased.
ANIMAL SCIENCE JOURNAL
(2022)
Article
Agriculture, Dairy & Animal Science
Zhao Namula, Manita Wittayarat, Lanh Thi Kim Do, Thanh Van Nguyen, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Takeshige Otoi
Summary: Electroporation before and after fertilization can achieve comparable efficiency in multiple gene editing, indicating that oocytes with completed maturation time may allow better functioning of materials accepting gene editing application.
VETERINARY AND ANIMAL SCIENCE
(2022)
Article
Agriculture, Dairy & Animal Science
Manita Wittayarat, Maki Hirata, Zhao Namula, Yoko Sato, Nhien T. Nguyen, Quynh A. Le, Qingyi Lin, Koki Takebayashi, Fuminori Tanihara, Takeshige Otoi
Summary: This study demonstrated the feasibility of introducing point mutations in porcine embryos through CRISPR/Cas9 delivery with ssODN via electroporation. The length of the ssODN homology arm significantly affected the efficiency of introducing point mutations. Further research is needed to improve the accuracy and efficiency of homology-directed repair.
ANIMAL SCIENCE JOURNAL
(2021)
Article
Agriculture, Dairy & Animal Science
Nhien Thi Nguyen, Manita Wittayarat, Zhao Namula, Yoko Sato, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Fuminori Tanihara, Maki Hirata, Takeshige Otoi
Summary: The study aimed to improve the embryonic development of blastocysts by supplementing antioxidants during in vitro maturation of mouse iPS cells. The combination of CGA and ITS increased total cell number in blastocysts, but had no effect on oocyte maturation rates. When mouse iPS cells were injected into porcine blastocysts, CGA supplementation showed benefits for interspecies chimera blastocyst development.
JOURNAL OF APPLIED ANIMAL RESEARCH
(2021)
Article
Agriculture, Dairy & Animal Science
Zhao Namula, Yasuhiro Isumi, Yoko Sato, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Chommanart Thongkittidilok, Takeshige Otoi
Summary: This study compared the quality and penetration ability of frozen-thawed spermatozoa from three microminipigs and Large White boars, and investigated the effects of caffeine and heparin, as well as sperm-oocyte co-incubation length on fertilization and embryonic development in vitro. Results showed differences in fertilization rates and developmental outcomes among the different boar breeds, with caffeine supplementation and longer co-incubation times showing beneficial effects for improving IVF results, especially with low-quality microminipig spermatozoa.
ARCHIVES ANIMAL BREEDING
(2021)
Article
Biology
Quynh Anh Le, Fuminori Tanihara, Manita Wittayarat, Zhao Namula, Yoko Sato, Qingyi Lin, Koki Takebayashi, Maki Hirata, Takeshige Otoi
Summary: Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are commonly used for generating genetically modified pigs, but they often lead to mosaic mutations in embryos. This study found that the gene editing method and embryonic stage for gene editing can significantly impact the gene editing efficiency and mutation rates of porcine embryos.
BMC RESEARCH NOTES
(2021)