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Animal Bioresource in Japan

.: Home > Animal Bioresource in Japan > 2014 > Volume 63 Number 3 > Yoshiko Nakagawa, Tetsushi Sakuma, Naomi Nakagata, Sho Yamasaki, Naoki Takeda, Masaki Ohmuraya, Takashi Yamamoto

Application of Oocyte Cryopreservation Technology in TALEN-Mediated Mouse Genome Editing

Yoshiko Nakagawa, Tetsushi Sakuma, Naomi Nakagata, Sho Yamasaki, Naoki Takeda, Masaki Ohmuraya, Takashi Yamamoto
1)Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan 2)Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan 3)Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Abstract :

Reproductive engineering techniques, such as in vitro fertilization (IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation, reproduction, and transportation of genetically engineered mice. However, it has not yet been elucidated whether these techniques can be applied for the generation of genome-edited mice using engineered nucleases such as transcription activator-like effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes fertilized in vitro using frozen sperm for TALEN-mediated genome editing in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen), fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the source of oocytes used for TALEN injection; we found that fertilized oocytes created under all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we investigated whether the ages in weeks of parental female mice can affect the efficiency of gene modification, by comparing 5-week-old and 8–12-week-old mice as the source of oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was consistently 95–100% when either 5-week-old or 8–12-week-old mice were used with or without freezing the oocytes. Thus, our report describes the availability of freeze-thawed oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome editing, thus boosting the convenience of such innovative gene targeting strategies.

Keywords :
Cryopreservation, Female age, Genome editing, Knockout mouse, TALEN

Date Deposited : 07 Apr 2015 10:32

Last Modified : 07 Apr 2015 10:32

Official URL: http://

Volume 63, Number 3, - 2014

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