Efficient generation of conditional knockout mice via sequential introduction of lox sites

Takuro Horii,Izuho Hatada,Mihiro Shibutani,Naomi Terawaki,Sumiyo Morita,Mika Kimura

doi:10.1038/s41598-017-08496-8

Takuro Horii, Izuho Hatada + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-08496-8

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Journal: Scientific Reports	Publication Date: Aug 11, 2017
Citations: 58	License type: open-access

Affiliation: Gunma University

Abstract

Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.

Highlights

According to the International Mouse Phenotyping Consortium, more than 60% (284/459) of knockout mouse strains (C57BL/6N background) show a prenatal lethality phenotype
We investigated simultaneous injection of Cas[9] protein, two sets of guide RNA (gRNA), and single-stranded oligodeoxynucleotides (ssODNs) including loxP variants lox[66] and lox7115 targeting the Mecp[2] locus using various concentrations of Cas9/gRNA/ ssODN (Fig. S1a)
We suspected that simultaneous injection of two gRNAs induced double-strand breaks (DSBs) at two sites on the same chromosome, which caused chromosomal deletion and reduced flox frequency

Summary

Introduction

According to the International Mouse Phenotyping Consortium (http://www.mousephenotype.org/), more than 60% (284/459) of knockout mouse strains (C57BL/6N background) show a prenatal lethality phenotype. Simultaneous injection of two sets of gRNAs and ssODNs including loxP sites generates mice containing floxed alleles at the Mecp[2] locus[11], but this can cause DSBs at two sites on the same chromosome, which can cause chromosomal deletions (Fig. 1a).

Results

Conclusion