RRC ID 35937
Author Mizuno S, Dinh TT, Kato K, Mizuno-Iijima S, Tanimoto Y, Daitoku Y, Hoshino Y, Ikawa M, Takahashi S, Sugiyama F, Yagami K.
Title Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.
Journal Mamm Genome
Abstract Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constructed a CRISPR/Cas9 expression vector for the Tyr gene (px330-Tyr-M). DNA cleavage activity of px330-Tyr-M at the target site of the Tyr gene was confirmed by the EGxxFP system. We also designed an ssDNA donor for homology-directed repair (HDR)-mediated gene modification. The px330-Tyr-M vector and ssDNA donor were co-microinjected into the pronuclei of 224 one-cell-stage embryos derived from C57BL/6J mice. We obtained 60 neonates, 28 of which showed the ocular albinism and absence of coat pigmentation. Genomic sequencing analysis of the albino mice revealed that the target of SNM, G291T in the Tyr gene, occurred in 11 mice and one founder was homozygously mutated. The remaining albino founders without Tyr G291T mutation also possessed biallelic deletion and insertion mutants adjacent to the target site in the Tyr locus. Simple production of albino C57BL/6J mice was provided by C57BL/6J zygote microinjection with px330-Tyr-M DNA vector and mutant ssDNA (G291T in Tyr) donor. A combination of CRISPR/Cas9 vector and optional mutant ssDNA could be expected to efficiently produce novel SNM-induced mouse models for investigating human diseases.
Volume 25(7-8)
Pages 327-34
Published 2014-8-1
DOI 10.1007/s00335-014-9524-0
PMID 24879364
MeSH Alleles Animals Base Sequence CRISPR-Cas Systems / genetics* DNA / genetics Female Gene Targeting / methods* Genetic Vectors / metabolism Genome / genetics Humans Male Mice, Inbred C57BL Mice, Inbred ICR Mice, Transgenic Microinjections Molecular Sequence Data Monophenol Monooxygenase / genetics* Mutation / genetics* Pedigree Zygote / metabolism
IF 2.287
Times Cited 50
DNA material px330-Tyr-M (RDB13943) pCAG-EGxxFP-Tyr-M (RDB13944).