Building Cre Knockin Rat Lines Using CRISPR/Cas9

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1642)

Abstract

Conditional gene inactivation strategy helps researchers to study the gene functions that are critical in embryogenesis or in defined tissues of adulthood. The Cre/loxP system is widely used for conditional gene inactivation/activation in cells or organisms. Cre knockin animal lines are essential for gene expression or inactivation in a spatially and temporally restricted manner. However, to generate a Cre knockin line by traditional approach is laborious. Recently, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has been proven as a simple and efficient genome-editing tool. We have used CRISPR/Cas9 system to generate rat strains that carry Cre genes in different targeted gene loci by direct delivery of gRNAs/Cas9/donors into fertilized eggs. Here, we described a stepwise procedure for the generation of Cre knockin rat, including target site selection, RNA preparation, the construction of the template donor, pronuclear injection, and the genotyping of precise Cre insertion in F0 rats. Taken together, the establishment of Cre knockin line can be achieved within 6 weeks.

Key words

Rat CRISPR/Cas9 Cre/loxP Homologous recombination Conditional knockout 

Notes

Acknowledgments

We thank the entire Huang laboratory and Zhang laboratory for their support and efforts on optimizing the CRISPR/Cas9 applications. This work was partially supported by the National Natural Science Foundation of China (31501001 and 31471400) and National Science and Technology Support Project (2014BAI02B01).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Key Laboratory of Human Disease Comparative Medicine, NHFPC, Institute of Laboratory Animal SciencePeking Union Medicine College, Chinese Academy of Medical SciencesBeijingChina
  2. 2.School of Life Science and TechnologyShanghaiTech UniversityShanghaiChina

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