Site-Specific Recombinases pp 21-35

Part of the Methods in Molecular Biology book series (MIMB, volume 1642) | Cite as

Direct Generation of Conditional Alleles Using CRISPR/Cas9 in Mouse Zygotes

  • Colin E. J. Pritchard
  • Lona J. Kroese
  • Ivo J. Huijbers
Protocol

Abstract

Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes. Previously, conditional alleles were introduced in the mouse germline by classic gene targeting in embryonic stem cells, a challenging and time-consuming procedure. Now, conditional alleles can be generated directly in fertilized mouse eggs (zygotes) using the CRISPR/Cas9 technology. This one-step generation of mice is easier in design and faster. Here, we describe our achieved success rate, the considerations in design of a conditional allele, a detailed protocol to prepare the zygote injection mix, and the screening procedure to identify the new conditional knockout mouse strain.

Key words

Transgenesis Conditional allele LoxP Cre recombinase CRISPR/Cas9 In vitro transcription Zygote Germline 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Colin E. J. Pritchard
    • 1
  • Lona J. Kroese
    • 1
  • Ivo J. Huijbers
    • 1
  1. 1.Mouse Clinic for Cancer and Aging—Transgenic Facility, The Netherlands Cancer InstituteAmsterdamThe Netherlands

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