Applications for Mu transposition technology

Mu can be used both for in vitro and in vivo applications

Mu in vitro transposition technology has been used for DNA sequencing, protein engineering for structure/function studies, genome-wide functional mapping of virus genomes, construction of gene targeting vectors, insertional mutagenesis of archaea, and SNP discovery. In addition, functional transpososomes can be pre-assembled in vitro and subsequently transformed into host cells, where DNA of interest can be transposed in vivo into the genome of the recipient cell. This combination of in vitro and in vivo systems can be used for highly efficient, species-non-specific gene delivery and insertional mutagenesis, as demonstrated with a variety of Gram-negative and Gram-positive bacteria, yeast, and mammalian cells.

General protocols and principles applicable for the Mu in vitro transposition reaction can be found here: Applications of the Bacteriophage Mu In Vitro Transposition Reaction and Genome Manipulation via Electroporation of DNA Transposition Complexes, Haapa-Paananen S, Savilahti H. Methods Mol Biol. 2018;1681:279-286. 

Scheme for in vitro transposition integration.  A tetramer of MuA transposase and Mu transposon ends assemble into a stable transpososome. Under reaction conditions with Mg2+, the transpososome becomes activated and executes transposon integration into the target DNA.

Scheme for genomic integration. A tetramer of MuA transposase and Mu transposon ends assemble into stable transpososomes in vitro. Following electroporation, the transpososomes encounter Mg2+ ions in vivo and integrate transposon DNA into the chromosome.

Table. Applications for Mu in vitro transposition technology

Use Application References
DNA sequencing Creation of sequencing templates Haapa et al. 1999, Brady et al. 2011

Rare mutation detection by next-generation DNA sequencing (NGS)

 Unique MuA-based Molec-

ular Indexing (UMAMI)

 Mielinis et al. 2021
Functional and structural studies of proteins Intein-assisted bisection mapping (IBM) method Ho et al. 2021
Bisection mapping Segall-Shapiro et al. 2014
Hexahistidine insertions Hoeller et al. 2008
Creating libraries of circularly

permuted proteins

 Mehta et al. 2012, Zeng et al. 2018
Creating libraries that express fragmented protein variants Segall-Shapiro et al. 2011
Pentapeptide insertion mutagenesis strategy Poussu et al. 2004, Pajunen et al. 2009
Gene truncation strategy Poussu et al. 2005
Triplet nucleotide removal method Jones et al. 2005, Simm et al. 2007
Single amino acid substitutions Baldwin et al. 2008, Dagget et al. 2009
Domain insertion strategy Edwards et al. 2008, Nadler et al. 2016, Oakes et al. 2016, Chai et al. 2024
Functional genetics and genomics of viruses Whole genome analysis of bacteriophages Vilen et al. 2003, Kiljunen et al. 2005, Krupovic et al. 2006,
Analysis of genomic regions and entire genomes of viruses cloned on specific vectors Laurent et al. 2000, Kekarainen et al. 2002
Construction of different kinds of gene targeting vectors Generation of null, hypomorphic, or conditional alleles Jukkola et al. 2005, Turakainen et al. 2009, Vilen et al. 2003, Zhang et al. 2005
Mapping single nucleotide polymorphism (SNP) Mismatch targeting Yanagihara and Mizuuchi 2002, Orsini et al. 2007
Identification of non-essential archaeal genes Generation of random genomic insertion mutant library for archaea Kiljunen et al. 2014, Legerme et al. 2016
Species non-specific gene delivery and insertional mutagenesis Use of in vitro pre-assembled transpososomes for gene delivery in vivo Lamberg et al. 2002, Lanckriet et al. 2009, Pajunen et al. 2005, Paatero et al. 2008, Tu Quoc et al. 2007, Wu et al. 2009
Cloning of circular DNA In vitro insertion of a transposon containing  an E.coli origin of replication Pulkkinen et al. 2016