Stabilizing Integrated Transgenes

CSIRO_ScienceImage_10746_An_adult_silkworm_mothWang et al (2015) describe their system for stabilizing transgenes inserted into the genome of Bombyx mori in a recent volume of Molecular Genetics and Genomics.

Having multiple transposon-based gene vector platforms, all of which work efficiently in a species can be very useful. For example, one might use a second vector system if one wanted to insert (stack) another transgene into an existing transgenic line. One might want to avoid using the original vector system since there would be the risk of remobilizing the initial insert to a new location or of loosing it altogether through excision.

But if you have only one transposon-based gene vector platform it is still possible to sequentially stack transgenes by stabilizing each following their integration. This can be useful.

Transposon

Cartoon of a transposon with transposase interacting with the inverted terminal repeats.

Furthermore, post-integration stabilization of transgenes might mitigate risks associated with altering or loosing a genotype/phenotype following a release of transgenic insects into the environment or when using transgenic insects for some other application.

Wang et al (2015) describe a system they introduce into Bombyx mori that results in a stably integrated transgene that is no longer associated with a functional transposon. Wang et al.’s system resembles the system described by Handler et al (2004) but with a slight modification.

The basic strategy involved creating a piggyBac element with a single right-inverted repeat (R1) but with two left-inverted repeats. The internal left-inverted repeat (L2) flanked a simple marker gene and the second (or ‘outside’) left-inverted repeat (L1) flanked the transgene of interest to be inserted and stabilized.

Stability2

The basic concept behind stabilizing a transposon gene vector. Element L1/R1 is integrated followed by the excision of element L2/R1, leaving the transgene in a ‘broken’ vector that can not be remobilized even in the presence of transposase.

The idea is to get the entire L1/R1 element to integrate and then, either simultaneously or at some later time, stimulate L2/R1 excision. This will result in the transgene being left in the genome with only L1 attached to it, and without R1 this becomes a broken vector incapable of further movement even in the presence of functional piggyBac transposase.

It is pretty well established that smaller transposons have higher rates of movement compared to longer transposons, everything else being equal. This means that L1/R1 transpositions will be less frequent than L2/R1 transpositions, making it challenging to get the transgene integrated initially. While Wang et al (2015) should be consulted for the details, in short, what they did was alter L2 so that it was somewhat less efficient as an inverted terminal repeat compared to L1.

This clever adjustment made the system somewhat more convenient and manageable, allowing transgene integration and subsequent stabilization to be more readily achieved.  Basically, their modification resulted in more L1/R1 transpositions relative to L2/R1 transpositions.   See the paper for details regarding what the altered L1 looked like.

This is another demonstration of transposon stabilization and it could be used in any insect system in which piggyBac transposons are capable of post-integration remobilization.

 

Wang, F, Wang, R, Wang, Y, Xu, H, Yuan, L, Ding, H, Ma, S, Zhou, Y, Zhao, P, Xia, Q. Remobilizing deleted piggyBac vector post-integration for transgene stability in silkworm. Mol. Genet. Genomics 2015; 290:1181-1189  doi: 10.1007/s00438-014-0982-6

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