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Persistence Pays: piggyBac Remobilization in Anopheles

October 13, 2011 - 11:52 -- Mark Benedict

Dave O’brochta of the University of Maryland has long insisted that merely developing the capacity to perform germline transformation of mosquitoes is not enough to claim a full-blown forward-genetic manipulation system. However, once transposable elements and vectors were available for Aedes and Anopheles, the enthusiasm for developing fundamentally new information about germline transformation waned – at least for many who would use it. Not for all though.

 

Persistence paid with discovery of an important difference between Aedes and Anopheles. O’brochta et al. (available free – thanks!) have uncovered a significant difference that is relevant to the field of genetic control of mosquitoes: the piggyBac transposable element that could be not be remobilized in Aedes aegypti CAN be in Anopheles stephensi. Not only does this expand one’s ability to move elements to new locations, remobilization provides a method to detect tissue- and stage-specific enhancers.

 

To provide a bit of background, scientists developing transgenesis in mosquitoes in the early days mimicked what was being done in Drosophila melanogaster, the birthplace of insect transgenesis. The behavior of the type II transposable “P element” was the main reference point. Insertions of this transposable element in the genome could be “re-mobilized” by providing the essential protein, transposase, “in trans” by injecting it into embryos or crossing to a strain that expressed it. This was a powerful means to greatly increase the ability to create novel insertions.

 

The potential for such remobilization worried those planning to use transposable elements to modify mosquitoes in nature. What if their transgene encountered a source of transposase that remobilized it? While the specific effects that might result were never carefully described, it was a biosafety concern that was always raised.

 

When piggyBac transformation of mosquitoes was developed, it was natural to assume that re-mobilization, like P, was possible. So much for assumptions. In spite of well designed experiments – an international effort including the O’brochta lab - conclusively demonstrated that piggyBac simply could not be remobilized in Aedes aegypti: a disappointing outcome for advanced genetic manipulation, a tentative sigh of relief for biosafety concerns. Mysterious outcome, but arguably a negative result that many would not want to test again. Fortunately, some would.

 

Fast forward four years. O’brochta et al. have demonstrated that piggyBac can be remobilized at high frequencies (~10%) in Anopheles stephensi. They detected numerous interesting modifications of fluorescent protein markers among the progeny – insertions affected by enhancers that control gene expression. (If you don’t read the article, you should at least view the images.) This observation underlines important distinctions between Aedes and Anopheles.

 

(Having been burned once on assumptions in this area, I’ll avoid suggesting this difference extends to other Aedes and Anopheles species. Nah! This is a blog. I’ll bet it’s a general characteristic of the genera.)

 

These observations provide important information about remobilization that will be interesting to determine in Anopheles gambiae. I’d be surprised if they (or another group) are not testing it already. It also revives concerns about stability of piggyBac elements in mosquitoes used in future release programs. While a specific interaction between transgenic mosquitoes carrying a piggyBac element and trans-mobilizing transposase may not exist AND a plausible harm that would result must result, the concern is back on the table. Certainly, it makes disabling insertions by removal of the essential terminal repeats worth considering as a standard biosafety measure in Anopheles destined for release.

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