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

被引：4

作者：

Nishizono, Hirofumi ^{[1
,2
,3
]}

Darwish, Mohamed ^{[4
,5
]}

Uosaki, Hideki ^{[6
,7
]}

Masuyama, Nanami ^{[8
,9
,10
]}

Seki, Motoaki ^{[8
,11
]}

Abe, Hiroyuki ^{[3
]}

Yachie, Nozomu ^{[8
,9
,10
,12
,13
]}

Yasuda, Ryohei ^{[1
]}

机构：

[1] Max Planck Florida Inst Neurosci, Jupiter, FL 33458 USA

[2] Univ Toyama, Life Sci Res Ctr, Toyama, Japan

[3] Yamagata Univ, Grad Sch Sci & Engn, Dept Biochem Engn, Yamagata, Japan

[4] Univ Toyama, Grad Sch Innovat Life Sci, Toyama, Japan

[5] Cairo Univ, Fac Pharm, Dept Biochem, Giza, Egypt

[6] Jichi Med Univ, Ctr Mol Med, Div Regenerat Med, Shimotsuke, Tochigi, Japan

[7] Jichi Med Univ, Ctr Dev Adv Med Technol, Div Stem Cell Res & Drug Dev, Shimotsuke, Tochigi, Japan

[8] Univ Tokyo, Res Ctr Adv Sci & Technol, Synthet Biol Div, Tokyo, Japan

[9] Keio Univ, Inst Adv Biosci, Tokyo, Japan

[10] Keio Univ, Grad Sch Media & Governance, Tokyo, Japan

[11] Chiba Univ, Grad Sch Med, Dept Mol Oncol, Chiba, Japan

[12] Univ Tokyo, Sch Sci, Dept Biol Sci, Tokyo, Japan

[13] Univ Tokyo, Coll Arts & Sci, Tokyo, Japan

来源：

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | 2020年 / 158期

关键词：

Genetics; Issue; 158; CRISPR/Cas9; one-cell embryo cryopreservation; electroporation; genetically modified mice; GM; genome editing; FBS; ELECTROPORATION; RNA; GENERATION; DELIVERY; OOCYTES; ZYGOTES; KNOCKIN;

D O I：

10.3791/60808

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：9

共 35 条

[1] CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection [J].