Advantages of transposon tagging in <I>Arabidopsis

Avoid unintentional mutagenesis. When a T-DNA (with or without a transposon) is introduced into Arabidopsis, mutations arise in the resulting lines. However, when transposons are used, mutagenesis can be separated from the transformation process that introduces the mutagen. This is because the transposase and the transposon T-DNAs are transformed into different plants. Transposition occurs only after the transposon line is crossed by the transposase line.

Get stable mutations that will revert. A mutation caused by a transposition-defective transposon is stable as long as the plant does not contain an active transposase gene. The transposon can be remobilized by reintroducing the transposase gene by a genetic cross. Revertants arise by excision of the transposon from the mutant gene. When the a plant transposon inserts, it creates a short duplication. Transposons of the Ac-Ds family create an 8-bp duplication. When the transposon excises, it leaves behind either the entire duplication or some part of it, often with a few base pairs deleted or added at the former insertion site. If the insertion was in an exon, the reading frame must be restored to obtain a revertant, so revertants of exon insertions generally have some multiple of a 3-bp insertion left behind. Detecting this so-called "footprint" proves that the mutation was caused by the insertion.

Get new mutations. If a transposon is inserted in an exon, excision of the transposon can change the sequence at the insertion site, resulting in a change, insertion or deletion of one to several amino acids. Changes in the activity of the gene caused by such mutations can provide insight into protein function.

Target your gene for insertion. Transposons of the Ac family have a marked preference for short-range transposition. This can greatly reduce the amount of work involved in identifying an insertion in a target gene if there is a closely linked transposon which can be mobilized.

Pick the right tagging system. Transposon tagging systems for Arabidopsis have been developed in several laboratories (see reference list). The efficiency of transposon tagging has been improved by using transposons that 1) are immobile and maintained in separate lines from the transposase, 2) carry selectable markers, and 3) disrupt a selectable marker gene whose function is restored upon excision, providing a means of identifying plants in which transposition has occurred. The source of transposase is either an immobile Ac element or cDNA for the element-encoded ORFA protein. Recent transposons carry reporter genes which are activated upon insertion into a gene or promoter, facilitating analysis of the gene's expression pattern.