- Transposons are mobile genetic elements that move (transpose) from one region of the genome to another.
- Transposons are used as tools for gene cloning because insertion of a transposon into a gene disrupts its function and produce a visible mutant phenotype.
- When the DNA sequence of the transposon is known, it is possible to clone the disrupted gene by using the transposable element as a “tag” to identify the segment of DNA harboring the element.
- Transposon tagging involves inducing transposition, screening for mutations caused by transposon insertion, identifying the element causing the mutation, and cloning the tagged gene.
- Transposons are found in almost all organisms where they have been looked for, including bacteria, yeast, plants, and mammals.
- Transposons from one organism often also transpose in heterologous systems.
- Therefore it is possible to use transposon-tagging methods to clone genes in a wide variety of organisms, including those where there are few other tools available for gene isolation.
Class II elements have inverted repeats at their ends and produce the products needed for their own excision and insertion, termed transposase.
Obtaining the Sequence of the Transposon “Tag”
- To use a transposon as a molecular “tag” for gene cloning, it is necessary to use a transposon with a known DNA sequence.
- Many transposons have already been well characterized.
- New transposons can be “trapped” by first mobilizing transposition and then identifying transposon insertions into known genes (generally by looking for unstable alleles of the gene).
- Then the mutant allele is sequenced, thus determining the sequence of the inserted transposon.
Transposon and Transposase Engineering
- It is useful to have transposons designed so that they carry selectable markers and they carry part of a plasmid that can be selected for in E. coli to facilitate cloning of flanking DNA by plasmid rescue.
- A marker linked to the transposase source facilitates removal of the transposase later by segregation from the tagged gene.
- It is also sometimes possible to increase transposition frequencies by deleting or altering part of the transposase gene, by using a powerful constitutive promoter to drive transposase, or by altering transposon size.