Tandem Recombineering by SLIC Cloning and Cre-LoxP Fusion to Generate Multigene Expression Constructs for Protein Complex Research
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 wordsSequence and ligation independent cloning Cre recombinase Cre-LoxP fusion Multigene delivery Multiprotein complexes MultiBac ACEMBL automation Robotics
We thank all members of the Berger laboratory for helpful discussions. M.H. is recipient of a Kekulé fellowship of the Fonds der Chemischen Industrie (FCI, Germany). Y.N. is a fellow of the Marie-Curie training network Chromatin Plasticity and the Boehringer Ingelheim Foundation (BIF, Germany). I.B. acknowledges support from the Swiss National Science Foundation (SNSF), the Agence Nationale de la Recherche (ANR), the Centre National de la Recherche Scientifique (CNRS), the EMBL and the European Commission (EC) through the joint EIPOD program, and the European Commission (EC) projects SPINE2-Complexes and 3D-Repertoire (Framework Program 6 (FP6)), as well as INSTRUCT, PCUBE, BioSTRUCT-X, and ComplexINC (EC FP7).
Competing financial interest statement: The authors declare competing financial interests. I.B. is author on patents and patent applications related to the methods here described.
- 7.Nie Y, Bieniossek C, Frey D, Olieric N, Schaffitzel C, Steinmetz MO, Berger I (2009) ACEMBLing multigene expression vectors by recombineering. Nat Protoc 4. doi: 10.1038/nprot.2009.104
- 8.Kriz A, Schmid K, Baumgartner N, Ziegler U, Berger I, Ballmer-Hofer K, Berger P (2010) A plasmid-based multigene expression system for mammalian cells. Nat Commun 1. doi: 10.1038/ncomms1120
- 11.Vijayachandran LS, Viola C, Garzoni F, Trowitzsch S, Bieniossek C, Chaillet M, Schaffitzel C, Busso D, Romier C, Poterszman A, Richmond TJ, Berger I (2011) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J Struct Biol 175:198–208CrossRefGoogle Scholar