Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization

• Published in: Scientific reports 2019-08, Vol.9 (1), p.11571-11571, Article 11571
• Main Authors: Honda, Arata, Tachibana, Ryoma, Hamada, Kazuya, Morita, Kohtaro, Mizuno, Naoaki, Morita, Kento, Asano, Masahide
• Format: Article
• Language: English
• Subjects: 13; 42/41; 45; 631/1647/334/1874/486; 631/208/135; 631/61/17/1511; 64; Anesthesia; Animals; CRISPR-Cas Systems; Electroporation; Electroporation - methods; Electroporation - veterinary; Embryos; Female; Fertilization in Vitro - methods; Fertilization in Vitro - veterinary; Gene Editing - methods; Gene Editing - veterinary; Gene Knock-In Techniques - methods; Gene Knock-In Techniques - veterinary; Gene Knockout Techniques - methods; Gene Knockout Techniques - veterinary; Genome editing; Genomes; Humanities and Social Sciences; In vitro fertilization; Inhibin; Male; multidisciplinary; Offspring; Oocytes; Ovulation; Rats - embryology; Rats - genetics; Rats - physiology; Rats, Inbred F344 - embryology; Rats, Inbred F344 - genetics; Rats, Inbred F344 - physiology; Rats, Long-Evans - embryology; Rats, Long-Evans - genetics; Rats, Long-Evans - physiology; Rats, Sprague-Dawley - embryology; Rats, Sprague-Dawley - genetics; Rats, Sprague-Dawley - physiology; Rats, Wistar - embryology; Rats, Wistar - genetics; Rats, Wistar - physiology; Rodents; Science; Science (multidisciplinary); Superovulation; Tyrosinase gene
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-47964-1

Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3–47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.