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Methods Mol Biol

Title:		Gene Targeting in Rabbits: Single-Step Generation of Knock-out Rabbits by Microinjection of CRISPR/Cas9 Plasmids
Author(s):		Kawano Y; Honda A;
Address:		"Organization for Promotion of Tenure Track, University of Miyazaki, 5200, Kibara, Kiyotake, Miyazaki, 889-1692, Japan. Organization for Promotion of Tenure Track, University of Miyazaki, 5200, Kibara, Kiyotake, Miyazaki, 889-1692, Japan. a-honda@med.miyazaki-u.ac.jp"
Journal Title:		Methods Mol Biol
Year:		2017
Volume:		1630
Issue:
Page Number:		109 - 120
DOI:		10.1007/978-1-4939-7128-2_10
ISSN/ISBN:		1940-6029 (Electronic) 1064-3745 (Linking)
Abstract:		"The development of genome editing technology has allowed gene disruptions to be achieved in various animal species and has been beneficial to many mammals. Gene disruption using pluripotent stem cells is difficult to achieve in rabbits, but thanks to advances in genome editing technology, a number of gene disruptions have been conducted. This paper describes a simple and easy method for carrying out gene disruptions in rabbits using CRISPR/Cas9 in which the gene to be disrupted is marked, the presence or absence of off-target candidates is checked, and a plasmid allowing simultaneous expression of Cas9 and sgRNA is constructed. Next, the cleaving activity of candidate sequences is investigated, and assessments are carried out to determine whether the target sequences can be cut. Female rabbits subjected to superovulation treatment are mated with male rabbits and fertilized eggs are collected, and then pronuclear injection of plasmid DNA is performed. The next day, the two-cell stage embryos are transplanted into pseudopregnant rabbits, and offspring are born within approximately 29-30 days. The genomic DNA of the offspring is then examined to check what types of genetic modifications have occurred. With the advent of CRISPR/Cas9, the accessibility of gene disruptions in rabbits has improved remarkably. This paper summarizes specifically how to carry out gene disruptions in rabbits"
Keywords:		Animals *CRISPR-Cas Systems Female Gene Knockout Techniques Gene Targeting/*methods/veterinary Genetic Vectors/administration & dosage Microinjections/*methods/veterinary Plasmids/genetics Rabbits Zygote/growth & development CRISPR/Cas9 Gene targeting Gen;
Notes:		"MedlineKawano, Yoshihiro Honda, Arata eng Research Support, Non-U.S. Gov't 2017/06/24 Methods Mol Biol. 2017; 1630:109-120. doi: 10.1007/978-1-4939-7128-2_10"