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Sci Rep. 2014 Mar 28;4:4513. doi: 10.1038/srep04513.

Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.

Author information

1
1] Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan [2].
2
1] Division of Developmental Biotechnology, Department of Bioscience and Genetics Research Institute, National Cerebral and Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita Osaka 565-8565, Japan [2].
3
1] Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan [2] Department of Laboratory Sciences, Graduate School of Health Sciences, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8514, Japan [3] Department of Obstetrics and Gynecology, Gunma CHUO General Hospital, 1-7-13, Kouun-cho, Maebashi, Gunma 371-0025, Japan.
4
Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan.
5
Department of Obstetrics and Gynecology, Gunma CHUO General Hospital, 1-7-13, Kouun-cho, Maebashi, Gunma 371-0025, Japan.
6
Department of Laboratory Sciences, Graduate School of Health Sciences, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8514, Japan.

Abstract

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.

PMID:
24675426
PMCID:
PMC5380110
DOI:
10.1038/srep04513
[Indexed for MEDLINE]
Free PMC Article
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