Journal
BIOLOGY OPEN
Volume 5, Issue 8, Pages 1142-1148Publisher
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/bio.019349
Keywords
Knockout; Floxed; CRISPR-Cas9; Zygote; Ultra-superovulation; Inhibin antiserum and equine chorionic gonadotropin (IASe)
Categories
Funding
- Japan Society for the Promotion of Science (JSPS) [15H04606, 16K18478]
- Grants-in-Aid for Scientific Research [26660112, 15H04606, 26293174, 16K18478, 15K07051, 26290070] Funding Source: KAKEN
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Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.
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