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Genetic Manipulations Using Cre and Mutant LoxP Sites

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Book cover Controlled Genetic Manipulations

Part of the book series: Neuromethods ((NM,volume 65))

Abstract

The bacteriophage P1-derived Cre/lox recombination system has been extensively used to engineer the genome of cultured cells and experimental animals. Cre recombinase recognizes the loxP site, which is composed of two 13-bp inverted repeats and an 8-bp spacer region, and mediates both intramolecular (excisive) and intermolecular (integrative) recombination between two loxP sites. The excision reaction is efficient and can be used in conditional knockout strategies. On the other hand, integrative recombination is inefficient because the integrated DNA retains loxP sites at both ends and is easily excised again if the Cre recombinase is still present. However, integrative recombination is expected to be a powerful tool for genome engineering in mouse embryonic stem (ES) cells because it allows precise and repeated knock-in of any DNA into lox sites placed in the genome. To promote integrative recombination, two kinds of mutant lox systems have been developed and successfully used in ES cells to produce exchangeable (multipurpose) alleles. In this chapter, we describe a Cre/mutant lox system for integrative recombination, and we present an application of this system to gene targeting. By incorporating mutant lox sites into gene targeting vectors, we can first produce a null allele. Subsequently, any gene of interest, including the Cre recombinase gene itself, fluorescent genes, luciferase genes, mutated cDNAs, and human cDNAs, can be inserted and expressed under the endogenous promoter of the targeted gene. By combining other recombination systems, such as Flp/FRT, we can also convert null alleles into conditional alleles.

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Author information

Authors and Affiliations

  1. Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan

    Kimi Araki & Ken-ichi Yamamura

  2. Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan

    Ken-ichi Yamamura

Authors
  1. Kimi Araki
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  2. Ken-ichi Yamamura
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Corresponding author

Correspondence to Ken-ichi Yamamura .

Editor information

Editors and Affiliations

  1. National Institute of Mental Health, National Institutes of Health, Convent Drive 35, Bethesda, 20892, Maryland, USA

    Alexei Morozov

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Araki, K., Yamamura, Ki. (2012). Genetic Manipulations Using Cre and Mutant LoxP Sites. In: Morozov, A. (eds) Controlled Genetic Manipulations. Neuromethods, vol 65. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-533-6_2

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  • DOI: https://doi.org/10.1007/978-1-61779-533-6_2

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-532-9

  • Online ISBN: 978-1-61779-533-6

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