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

Hirofumi Nishizono; Mohamed Darwish; Hideki Uosaki; Nanami Masuyama; Motoaki Seki; Hiroyuki Abe; Nozomu Yachie; Ryohei Yasuda

doi:10.3791/60808

JoVE Journal
Genetics

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JoVE Journal Genetics

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

유전자 변형 마우스의 고효율 생산을 위한 동결 해동 배아 사용

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06:46 min

April 2, 2020

DOI: 10.3791/60808-v

Hirofumi Nishizono*^1,2,3, Mohamed Darwish*^4,5, Hideki Uosaki^6,7, Nanami Masuyama^8,9,10, Motoaki Seki^8,11, Hiroyuki Abe³, Nozomu Yachie^8,9,10,12,13, Ryohei Yasuda¹

¹Max Planck Florida Institute for Neuroscience, ²Life Science Research Center,University of Toyama, ³Department of Biochemical Engineering, Graduate School of Science and Engineering,Yamagata University, ⁴Graduate School of Innovative Life Science,University of Toyama, ⁵Department of Biochemistry, Faculty of Pharmacy,Cairo University, ⁶Division of Regenerative Medicine, Center for Molecular Medicine,Jichi Medical University, ⁷Division of Stem Cell Research and Drug Development, Center for Development of Advanced Medical Technology,Jichi Medical University, ⁸Synthetic Biology Division, Research Center for Advanced Science and Technology,University of Tokyo, ⁹Institute for Advanced Biosciences,Keio University, ¹⁰Graduate School of Media and Governance,Keio University, ¹¹Department of Molecular Oncology, Graduate School of Medicine,Chiba University, ¹²Department of Biological Sciences, School of Science,University of Tokyo, ¹³College of Arts and Sciences,University of Tokyo

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Overview

This article presents a modified method for cryopreservation of one-cell embryos and a protocol that combines freeze-thawed embryos with electroporation. This approach facilitates the efficient generation of genetically modified mice.

Key Study Components

Area of Science

Genetic modification
Embryo cryopreservation
Electroporation techniques

Background

Genetically modified mice are essential for various biological research.
Traditional methods of embryo manipulation can be inefficient.
Electroporation can enhance the efficiency of gene transfer.
Cryopreservation allows for the long-term storage of embryos.

Purpose of Study

To improve the efficiency of generating genetically modified mice.
To develop a reliable protocol for using cryopreserved embryos.
To reduce mosaicism in genetically modified offspring.

Methods Used

Super ovulation of female C57BL/6J mice using hormonal injections.
Collection and capacitation of spermatozoa from male mice.
In vitro fertilization of oocytes with capacitated sperm.
Application of electroporation to introduce genetic modifications.

Main Results

The protocol allows for rapid generation of genetically modified mice.
High efficiency and low mosaic rates were achieved.
Successful use of freeze-thawed embryos in the process.
Demonstrated feasibility of the method in a laboratory setting.

Conclusions

This method enhances the generation of genetically modified mice.
Combining cryopreservation with electroporation is effective.
The protocol can be a valuable tool for researchers in genetics.

Frequently Asked Questions

What is the main advantage of this protocol?

The protocol allows for efficient generation of genetically modified mice with reduced mosaicism.

Can this method be applied to other mouse strains?

While this study focuses on C57BL/6J mice, the method may be adaptable to other strains.

What are the key steps in the protocol?

Key steps include super ovulation, sperm collection, in vitro fertilization, and electroporation.

How does electroporation improve gene transfer?

Electroporation creates temporary pores in cell membranes, facilitating the uptake of genetic material.

Is cryopreservation necessary for this method?

Cryopreservation allows for the storage of embryos, making the process more flexible and efficient.

What are the implications of this research?

This research provides a streamlined approach for generating genetically modified models, aiding various biological studies.

여기에서, 우리는 1 세포 배아의 동결 보존을 위한 수정된 방법 뿐만 아니라 유전자 변형 마우스의 효율적인 생성을 위한 동결 해동 된 배아 및 전기 천공의 사용을 결합하는 프로토콜을 제시한다.

우리의 프로토콜은 단일 세포 마우스 배아에서 유전자 변형 마우스의 쉽고 효율적이며 신속하게 제조를 용이하게합니다. 우리의 프로토콜은 동결 해동 된 1 세포 단계 배아와 전기 기공의 사용을 결합하여 고효율 및 낮은 모자이크 속도로 돌연변이 마우스를 포함한 유전자 변형 마우스의 빠른 생성을 허용합니다. 체외 수정의 경우, 임신 한 암말 혈청 생식선 호르몬의 7.5 국제 단위의 인트라보네탈 주사를 통해 4 또는 8 주 된 여성 C57BL / 6J 마우스를 4 8 시간 후에 슈퍼 배란하여 시작하십시오.

다음으로, 성적으로 성숙한 3~5개월 된 남성 C57BL/6J 마우스에서 카우다 부피티미스를 제거하고 해부 바늘을 사용하여 정자의 혈전을 추출합니다. 그런 다음 HTF의 200 마이크로 리터 드롭에서 혈전을 섭씨 37도, 이산화탄소 는 5%로 1.5시간 동안 배양합니다. 인간의 융기 생식선 주입 후 16~18시간, 난소에서 적정 성 난낭 복합체를 수집하고 200개의 HTF 마이크로리터로 복합체를 배양하여 세포 배양 인큐베이터에서 2시간 이하의 배양에 대한 큐베이션을 한다.

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