Successful <i>i</i>-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein by Shuji Takabayashi, Kenta Iijima, Masumi Tsujimura, Takuya Aoshima, Hisayoshi Takagi, Kazushi Aoto, Masahiro Sato

Subjects: Get full text

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ Cells, Embryos, and Fetuses in Mice by Masahiro Sato, Shuji Takabayashi, Eri Akasaka, Shingo Nakamura

Subjects: Get full text

Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups by Shingo Nakamura, Kazunori Morohoshi, Emi Inada, Yoko Sato, Satoshi Watanabe, Issei Saitoh, Masahiro Sato

“…Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining established lines. …”
Get full text

Recent Advances in the Production of Genome-Edited Rats by Masahiro Sato, Shingo Nakamura, Emi Inada, Shuji Takabayashi

“…In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. …”
Get full text

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice by Masahiro Sato, Rico Miyagasako, Shuji Takabayashi, Masato Ohtsuka, Izuho Hatada, Takuro Horii

“…Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. …”
Get full text

Recent Genome-Editing Approaches toward Post-Implanted Fetuses in Mice by Shingo Nakamura, Emi Inada, Issei Saitoh, Masahiro Sato

“…There are at least four ways to induce genome editing in individuals: the first is to perform genome editing at the early preimplantation stage, such as fertilized eggs (zygotes), for the creation of whole genetically modified animals; the second is at post-implanted stages, as exemplified by the mid-gestational stages (E9 to E15), for targeting specific cell populations through in utero injection of viral vectors carrying genome-editing components or that of nonviral vectors carrying genome-editing components and subsequent in utero electroporation; the third is at the mid-gestational stages, as exemplified by tail-vein injection of genome-editing components into the pregnant females through which the genome-editing components can be transmitted to fetal cells via a placenta-blood barrier; and the last is at the newborn or adult stage, as exemplified by facial or tail-vein injection of genome-editing components. …”
Get full text

Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium by Masahiro Sato, Nami Sato-Yamamoto, Ai Wakita, Misako Haraguchi, Manabu Shimonishi, Hiroyuki Okuno

“…Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system—a modern genome editing technology—AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. …”
Get full text

Modification of <i>i</i>-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain by Yukari Kobayashi, Takuya Aoshima, Ryota Ito, Ryota Shinmura, Masato Ohtsuka, Eri Akasaka, Masahiro Sato, Shuji Takabayashi

“…Improved genome editing via oviductal nucleic acid delivery (<i>i</i>-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. <i>i</i>-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). …”
Get full text