standard A Stable, Replaceable, Highly Efficient Transgene Expression System

Bombyx mori

Bombyx mori

Long et. al. in Scientific Reports describe a system for creating stable sites in the genome of Bombyx mori that allow for site-specific recombination-mediated integration of transgenes.

Transposons are essential platforms for many insect genetic technologies and are typically used to introduce transgenes into insect genomes. The piggyBac transposon is used extensively in the silkworm Bombyx mori for this purpose.

Basic idea behind Recombination Mediated Cassette Exchange. Image Credit: Wu Lab, Harvard U.

Basic idea behind Recombination Mediated Cassette Exchange.
Image Credit: Wu Lab, Harvard U.

For some applications of transposon –based technologies the risk of the integrated element subsequently excising and/or transposing needs to be mitigated. Removing access to piggyBac encoded transposase is the primary means for stabilizing integrated elements but in some species there are endogenous piggyBac-like elements and corresponding transposases that might interact with introduced elements, causing their unintended movement.

Bombyx has many piggyBac like elements although not seemingly capable of mobilizing introduced piggyBac elements.

This is a problem that has been addressed in the past and strategies for removing one of the essential terminal inverted repeats of the transposon have been described (see references in Long et al. ).

Recombinase mediated cassette exchange (RMCE) can be a good way to integrate transgenes into a common site, so long as the genomic context is favorable for gene expression, something that is often not extensively tested.

Scheme

Basic scheme described by Long et al. 2015

Long et al. come up with their own strategy but in their case they have a system that removes all piggyBac vector sequences, leaving behind a dominant visible marker gene flanked only by the attachment sites of the site-specific recombination.

Those recombination sites can be repeatedly used to replace the transgene using phiC31 recombinase, although that was not demonstrated in this study.

Previous efforts to make vectors that could be triggered to self-distruct and become immobile have relied on the use of piggyBac elements that have three copies of the element’s terminal respeats – say one left ITR and two copies of the right ITR. When configure properly these elements can choose one of two way to transpose, with the inside or outside right-hand ITR. Excision therefore can result in the remove of two ITRs and leaving only a single ITR in the genome which is not expected to be capable of moving.

Long et al.’s strategy is similar but they have two tandem piggyBac elements in opposite orientation with a third marker gene in between that is flanked by phiC31 attachment sites.

The tandem element can act as one long element, carrying each of the two elements and the intervening phiC31 recombination sites into the genome. Subsequent excision of each of the two elements (one of them is carrying piggyBac transposase regulated with a heat responsive promoter) leaves only a marker gene flanked by phiC31 att sites. This complete removal of all initial gene vector sequences mitigates all risks associated with transgene immobility due to ‘cross mobilization’ by related endogenous transposable elements.

One of the attP-containing lines created by Long et al. seems to be conducive to high levels of transgene expression and is expected to be a useful line for introducing other transgenes where high levels of gene expression are desirable.

There were a lot of genetic manipulations described in this study and the paper provides a nice opportunity to see the productive combination of various genetic technologies to create genotypes of some practical use.

Dingpei Long, Weijian Lu, Yuli Zhang, Lihui Bi, Zhonghuai Xiang & Aichun Zhao  (2015) An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.Scientific Reports 5, Article number: 8802 doi:10.1038/srep08802

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