title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.
abstract: Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation,in vitrofertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice.
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Nakagawa
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan tetsushi-sakuma@hiroshima-u.ac.jp.
authors.forename: Tetsushi
authors.identifier: http://orcid.org/0000-0003-0396-1563
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Cell-Matrix Frontier Laboratory, Health Administration Center, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8551, Japan.
authors.forename: Norihisa
authors.initials: N
authors.lastname: Nishimichi
authors.affiliation: Cell-Matrix Frontier Laboratory, Health Administration Center, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8551, Japan.|Clinical Genetics, Hiroshima University Hospital, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8551, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Yokosaki
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takeo
authors.affiliation: Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Naomi
authors.initials: N
authors.lastname: Nakagata
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
keywords: CRISPR-Cas9; Culture time; Knock-in; Microinjection; Single-stranded oligodeoxynucleotide (ssODN); Zygote