Tandem Recombineering by SLIC Cloning and Cre-LoxP Fusion to Generate Multigene Expression Constructs for Protein Complex Research

  • Matthias Haffke
  • Cristina Viola
  • Yan Nie
  • Imre Berger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1073)

Abstract

A robust protocol to generate recombinant DNA containing multigene expression cassettes by using sequence and ligation independent cloning (SLIC) followed by multiplasmid Cre-LoxP recombination in tandem for multiprotein complex research is described. The protocol includes polymerase chain reaction (PCR) amplification of the desired genes, seamless insertion into the target vector via SLIC, and Cre-LoxP recombination of specific donor and acceptor plasmid molecules, optionally in a robotic setup. This procedure, called tandem recombineering, has been implemented for multiprotein expression in E. coli and mammalian cells, and also for insect cells using a recombinant baculovirus.

Key words

Sequence and ligation independent cloning Cre recombinase Cre-LoxP fusion Multigene delivery Multiprotein complexes MultiBac ACEMBL automation Robotics 

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Matthias Haffke
    • 1
    • 2
  • Cristina Viola
    • 1
    • 2
  • Yan Nie
    • 1
    • 2
  • Imre Berger
    • 1
    • 2
  1. 1.European Molecular Biology Laboratory (EMBL), BP 181Polygone ScientifiqueGrenobleFrance
  2. 2.Unit of Virus Host Cell Interactions (UVHCI)Polygone ScientifiqueGrenobleFrance

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