title: The kick-in system: a novel rapid knock-in strategy.
abstract: Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a "kick-in" to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.
authors.affiliation: Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Tomonoh
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Deshimaru
authors.affiliation: Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan.
authors.forename: Kimi
authors.initials: K
authors.lastname: Araki
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Miyazaki
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Arasaki
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka, Japan.
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka, Japan.
authors.forename: Haruna
authors.initials: H
authors.lastname: Kitamura
authors.affiliation: Department of Neuropathology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Mori
authors.affiliation: Department of Neuropathology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Wakabayashi
authors.affiliation: Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Saito
authors.affiliation: Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Kodaira, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Itoh
authors.affiliation: Central Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Uchida
authors.affiliation: Department of Neurophysiology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Yamada
authors.affiliation: Department of Neurophysiology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Migita
authors.affiliation: Department of Neurophysiology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Ueno
authors.affiliation: Brain Research Institute, Niigata University, Niigata, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kitaura
authors.affiliation: Brain Research Institute, Niigata University, Niigata, Japan.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Kakita
authors.affiliation: Department of Neurology, School of Medicine, University of California Davis, Sacramento, California, United States of America.
authors.forename: Christoph
authors.initials: C
authors.lastname: Lossin
authors.affiliation: Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Takano
authors.affiliation: Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan ; Central Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Hirose
mesh_terms: D000818:Animals; D001522:Behavior, Animal; D053595:Embryonic Stem Cells / Q000378:metabolism; D020936:Epilepsy, Benign Neonatal / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques / Q000379:methods*; D006801:Humans; D051658:KCNQ2 Potassium Channel / Q000235:genetics*; D008297:Male; D051379:Mice; D009154:Mutation; D010433:Pentylenetetrazole / Q000009:adverse effects; D011247:Pregnancy; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D013997:Time Factors
chemical_list: D051658:KCNQ2 Potassium Channel; D016760:Proto-Oncogene Proteins c-fos; D010433:Pentylenetetrazole