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Developmental Biology

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CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

3.7K Views

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10:57 min

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August 24th, 2022

DOI :

10.3791/64385-v

August 24th, 2022

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Manabu Ozawa*¹, Chihiro Emori*², Masahito Ikawa¹^,²

¹Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, ²Research Institute for Microbial Diseases, Osaka University

Summary

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

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Embryonic Stem Cells

Genetically Modified Mice

Gene Manipulation

Medical Science

Mouse Embryonic Fibroblast

Electroporation

ESC Culture Medium

Mitotically Inactivated MEF

DNA Implantation

Phenotype Analysis

Chapters in this video

0:04

Introduction

1:06

Gene Targeting of Embryonic Stem Cells (ESCs)

4:16

PCR Genotyping of Targeted ESCs

7:02

Preparation of Eight-Cell Stage Embryo and Microinjection of ESCs

9:49

Results: Screening of Targeted ESC Clones

10:10

Conclusion

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