Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. crR.......

Discussion

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.......

Materials

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