F344 jcl

F344/Jcl (CLEA Japan, Inc. Tokyo, Japan) rat embryos were collected from 7 to 8 weeks of age females that were superovulated by administration of 150 U/kg of PMSG followed by 75 U/kg of HCG. After natural mating, pronuclear-stage embryos were collected from oviducts of the females and cultured in a modified Krebs–Ringer bicarbonate medium (ARK Resource, Kumamoto, Japan). For electroporation, 50–100 embryos 3–4 h after collecting were placed into the chamber with 40 µl of serum free media (Opti-MEM Thermo Fisher Scientific MA, USA) containing 400 ng/µl Cas9 mRNA, 200 ng/µl gRNA. They were electroporated with a 5 mm gap electrode (CUY505P5 or CUY520P5 Nepa Gene, Chiba, Japan) in a NEPA21 Super Electroporator (Nepa Gene, Chiba, Japan). The poring pulses for the electroporation were voltage 225 V, pulse width 2.0 ms for rat embryos, pulse interval 50 ms, and number of pulses + 4. The first and second transfer pulses were voltage 20 V, pulse width 50 ms, pulse interval 50 ms, and number of pulses + 5. Embryos that developed to the two-cell stage after the introduction of RNAs were transferred into the oviducts of female surrogates anesthetized with isoflurane (DS Pharma Animal Health Co., Ltd., Osaka, Japan).

Genome editing by CRISPR-Cas was performed as described previously (Yoshimi et al., 2016 (link)). Guide RNAs (gRNAs) were designed by Optimized CRISPR Design (crispr.mit.edu) and synthesized by Integrated DNA Technologies, Inc., (Coralville, IA, United States). Long ssODNs (lsODNs) were prepared using a LsODN Preparation Kit (Biodynamics Laboratory Inc., Tokyo, Japan). Cas9 protein was purchased from Integrated DNA Technologies. Pronuclear-stage embryos of F344/Jcl (CLEA Japan, Inc., Tokyo, Japan) rats were produced by natural mating. The oviducts of female rats with vaginal plugs were removed after euthanasia by CO₂ and cervical dislocation, and embryos were flushed out from the ampullae with culture medium. Cas9 protein, gRNAs, and lsODN were introduced into the embryos using a super electroporator NEPA 21 (NEPA GENE Co., Ltd., Ichikawa, Chiba, Japan). Embryos that developed to the two-cell stage were transferred into the oviducts of pseudopregnant females that were anesthetized using isoflurane. Offspring were genotyped by the Amp-FTA method with the following primer set: 5′-CGGGTTTCAGAGATGGAAGA-3′ and 5′-ATTTTCATTGACAGGTCCGG-3′ and Ampdirect Plus buffer (Shimadzu Corporation, Kyoto, Japan). Founder rats were mated with F344/Jcl rats and F1 heterozygous rats were intercrossed to obtain F2 progeny.