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,
Functional genetics and genomics of viruses	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