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Methods Mol Biol. 2020;2066:89-100. doi: 10.1007/978-1-4939-9837-1_7.

Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage Embryos.

Author information

1
Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada. bin.gu@sickkids.ca.
2
The Centre for Phenogenomics (TCP), Toronto, ON, Canada.
3
Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada.
4
Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

Abstract

Large fragment knock-in mouse models such as reporters and conditional mutant mice are important models for biological research. Here we describe 2-cell (2C)-homologous recombination (HR)-CRISPR, a highly efficient method to generate large fragment knock-in mouse models by CRISPR-based genome engineering. Using this method, knock-in founders can be generated routinely in a time frame of about two months with high germline transmission efficiency. 2C-HR-CRISPR will significantly promote the advancement of basic and translational research using genetic mouse models.

KEYWORDS:

2-Cell stage; CRISPR-Cas9; Homologous recombination; Knock-in

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