title: Application of oocyte cryopreservation technology in TALEN-mediated mouse genome editing.
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.
authors: Center for Animal Resources and Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
authors: Yoshiko
authors: Y
authors: Nakagawa
authors: Tetsushi
authors: T
authors: Sakuma
authors: Naomi
authors: N
authors: Nakagata
authors: Sho
authors: S
authors: Yamasaki
authors: Naoki
authors: N
authors: Takeda
authors: Masaki
authors: M
authors: Ohmuraya
authors: Takashi
authors: T
authors: Yamamoto
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D005307:Fertilization in Vitro; D018390:Gene Targeting / Q000379:methods*; D016678:Genome / Q000235:genetics*; D008297:Male; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D008845:Microinjections; D009865:Oocytes; D012099:Reproductive Techniques; D012260:Ribonucleases / Q000008:administration & dosage / Q000235:genetics*; D012662:Semen Preservation; D013094:Spermatozoa; D015533:Transcriptional Activation
chemical_list: D012260:Ribonucleases