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* These authors contributed equally
Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.
The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a scientific breakthrough that provides unprecedented targeted modification in the genome1. The CRISPR/Cas9 system is comprised of Cas9 protein and guide RNA (gRNA) with two molecular components: a target-specific CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA)2 . A gRNA directs the Cas9 protein to the specific locus in the genome, 20 nucleotides complementary to crRNA, and adjacent to the protospacer adjacent motif (PAM). The Cas9 protein binds to the target sequence and induces double-stra....
All animal care and procedures performed in this study were undertaken according to the rules and regulations of the Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Care Committee of Laboratory Animals of University of Toyama, University of Tokyo, Jichi University, and Max Planck Florida Institute for Neuroscience. Information about all reagents is showed in the Table of Materials.
1. CRISPR reagents design
Our modified method for cryopreservation of one-cell embryos, including incubation in HTF containing 20% FBS for 10 min followed by cryopreservation in 1 M DMSO and DAP213 solution, improved the developmental rate of the freeze-thawed embryos into the two-cell stage (Figure 1, p = 0.009, Student’s t-test).The freeze-thawed embryos were used for the production of GM mice and electroporation conditions were optimized: five repeats of 25 V with 3 ms pulses and 97 ms intervals using an ele.......
The described protocol allows for the generation of GM mice with high efficiency and low mosaic rates (Table 1). It enables researchers without advanced embryo manipulation skills to create mutant mice easily because it takes advantage of the latest and most useful advances in both reproductive engineering and genome editing technologies: CRISPR/Cas9 ribonucleoprotein (RNP) and electroporation into freeze-thawed embryos. These advances facilitated and expedited the generation of the GM mice. As described.......
We wish to thank Hitomi Sawada and Elizabeth Garcia for animal care. This work was supported by KAKENHI (15K20134, 17K11222, 16H06276 and 16K01946) and Hokugin Research Grant (to H.N.), and Jichi Medical University Young Investigator Award (to H.U.). The Otsuka Toshimi Scholarship Foundation supported M.D.
....Name | Company | Catalog Number | Comments |
0.25 M Sucrose | ARK Resource Co., Ltd. (Kumamoto, Japan) | SUCROSE | |
1 M DMSO | ARK Resource Co., Ltd. (Kumamoto, Japan) | 1M DMSO | |
Butorphanol | Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) | Vetorphale 5mg | |
Cas9 protein: Alt-R® S.p. HiFi Cas9 Nuclease 3NLS | Integrated DNA Technologies, Inc. (Coralville, IA) | 1081060 | |
C57BL/6J mice | Japan SLC (Hamamatsu, Japan) | N/A | |
DAP213 | ARK Resource Co., Ltd. (Kumamoto, Japan) | DAP213 | |
FBS | Sigma-Aldrich, Inc. (St. Louis, MO) | ES-009-C | |
hCG | MOCHIDA PHARMACEUTICAL CO., LTD (Tokyo, Japan) | HCG Mochida 3000 | |
HTF | ARK Resource Co., Ltd. (Kumamoto, Japan) | HTF | |
ICR mice | Japan SLC (Hamamatsu, Japan) | N/A | |
Isoflurane | Petterson Vet Supply, Inc. (Greeley, CO) | 07-893-1389 | |
KSOM | ARK Resource Co., Ltd. (Kumamoto, Japan) | KSOM | |
LN2 Tank | Chart Industries (Ball Ground, GA) | XC 34/18 | |
M2 | ARK Resource Co., Ltd. (Kumamoto, Japan) | M2 | |
Medetomidine | Nippon Zenyaku Kogyo Co.,Ltd. (Koriyama, Japan) | 1124401A1060 | |
Microscope | Nikon Co. (Tokyo, Japan) | SMZ745T | |
Midazolam | Sandoz K.K. (Tokyo, Japan) | 1124401A1060 | |
Nuclease free buffer | Integrated DNA Technologies, Inc. (Coralville, IA) | 1072570 | |
Nucleospin DNA extraction kit | Takara Bio Inc (Kusatsu, Japan) | 740952 .5 | |
One-hole slide glass | Matsunami Glass Ind., Ltd. (Kishiwada, Japan) | S339929 | |
One-step type Electroporator | BEX Co., Ltd. (Tokyo, Japan) | CUY21EDIT II | |
Paraffin Liquid | NACALAI TESQUE Inc. (Kyoto, Japan) | SP 26137-85 | |
Platinum plate electrode | BEX Co., Ltd. (Tokyo, Japan) | LF501PT1-10, GE-101 | |
PMSG | ASKA Animal Health Co., Ltd (Tokyo, Japan) | SEROTROPIN 1000 | |
Povidone iodide | Professional Disposables International, Inc. (Orangeburg, NY) | C12400 | |
Reduced-Serum Minimal Essential Medium: OptiMEM I | Sigma-Aldrich, Inc. (St. Louis, MO) | 22600134 | |
Two-step type Electroporator | Nepa Gene Co., Ltd. (Ichikawa, Japan) | NEPA21 |
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