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PLoS One. 2014 Feb 19;9(2):e88549. doi: 10.1371/journal.pone.0088549. eCollection 2014.

The kick-in system: a novel rapid knock-in strategy.

Author information

  • 1Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan.
  • 2Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka, Japan.
  • 3Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan.
  • 4Department of Neuropathology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
  • 5Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan.
  • 6Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Kodaira, Japan.
  • 7Central Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan.
  • 8Department of Neurophysiology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
  • 9Brain Research Institute, Niigata University, Niigata, Japan.
  • 10Department of Neurology, School of Medicine, University of California Davis, Sacramento, California, United States of America.
  • 11Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan ; Central Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan.

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.

PMID:
24586341
[PubMed - in process]
PMCID:
PMC3929540
Free PMC Article

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