Trangenic Mosquitos and Dengue fever.. part two

Genetic modification to produce RIDL

The RIDL trait was created using a transposon. Transposons are mobile genetic elements (‘jumping genes'). They are similar to viruses, but lack the ability to form viral coats. RIDL was created by the piggyBac transposon originally isolated from a culture of cells of the cabbage looper, and has been used extensively in insect genetic-engineering. The piggyBac vector is prevented from replicating independently of the chromosome bearing it (non-autonomous) by removing its transposase enzyme that enables it to multiply and move among the chromosomes of the cells that it infects (though this is by no means a safeguard, see below) .

The transgenic male mosquito to be released has incorporated a gene for a red fluorescent marker protein for easy identification, but the key gene that confers dominant lethal trait is tTAV , encoding a tetracycline repressible transcription activator protein, driven by the promoter tetO of a Drosophila heat shock protein gene. In the presence of tetracycline, tTAV binds tetracycline and the complex does not bind to tetO , so no further expression of tTAV takes place. In the absence of tetracycline, there is a positive feedback loop in which tTAV binds to tetO , driving more expression of tTAV. The over production of tTAV is toxic, and kills the insect. But it is uncertain why excessive tTAV is lethal. In summary, RIDL is a tetracycline-repressible lethal system. It has been suggested that the lethality of excessive transcription activator is due to transcriptional ‘squelching' or interference with ubiquitin-dependent breakdown of proteins. Mice modified with a gene for the tetracycline repressible transcription activator were not killed when the gene was activated by removing tetracycline.

Is this terminator insect safe?


The most glaring aspect of the proposed release is that the lethally acting transcription activator tTAV has a rather ill-defined action. The information presently available does not tell us what is killing the target animals. Even though a homologous tetracycline-repressed gene was not toxic to mice upon its activation, the killing toxin in the mosquito should certainly be identified before released to the environment is contemplated.

Another major hazard is horizontal gene transfer of the piggyBac insert. This issue has been thoroughly addressed in ISIS' submissions to the USDA with regard to the release of the pink bollworm in 2001. We provided evidence that the disabled vector carrying the transgene, even when stripped down to the bare minimum of the border repeats, was nevertheless able to replicate and spread, basically because the transposase function enabling the piggyBac inserts to move can be supplied by ‘helper' transposons. Such helper transposons are potentially present in all genomes, including that of the mosquito. The main reason for using transposons as vectors in insect control is precisely because they can spread the transgenes rapidly by ‘non-Mendelian' mean within a population, i.e., by replicating copies and jumping into genomes, including those of the mammalian hosts. Although each transposon has its own specific transposase enzyme that recognizes its terminal repeats, the enzyme can also interact with the terminal repeats of other transposons, and evidence suggest “extensive cross-talk among related but distinct transposon families” within a single eukaryotic genome.

It is disingenuous to claim that because only male mosquitoes are released that don't bite people or other mammals, the technique is “environmentally benign”. First of all, the transgenic mosquitoes, both males and females, have to be mass-produced in the laboratory. In order for transgenic females, also carrying the dominant lethal in double dose, to propagate the line, they have to take blood meals from laboratory animals such as mice or rabbits, not to mention the odd lab worker, which gives plenty of opportunity for horizontal gene transfer. Second, the transgenic males have to be sorted from the females, and this takes place at the pupae stage, when males are generally smaller than females, but this may not be 100 percent accurate. Third, the tetracycline-dependence of the transgenic lines is not absolute. In the absence of tetracycline, 3 to 4 percent of transgenic progeny actually survive to adulthood.

It is obvious that transgene escape can readily occur. As Ho commented: “ These artificial transposons are already aggressive genome invaders, and putting them into insects is to give them wings, as well as sharp mouthparts for efficient delivery to all plants and animals and their viruses.”

One cannot stress enough that horizontal gene transfer and recombination is the main highway to exotic disease agents.

The piggyBac inserts may also be mobilised by the transposase of piggyBac transposons already carried by Baculovirus (a common soil-borne insect virus) that infect insect cells, and this possibility has not been evaluated in the laboratory. Baculovirus not only carries piggyBac transposons, it has also been used in human gene therapy as it is capable of infecting human cells. It is indeed strange that the mobility and horizontal gene transfer of the piggyBac vector has not been thoroughly studied even though the activity of the vector is widely recognized.

The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia , the cabbage looper, where it causes high mutation rates in the Baculovirus infecting the cells by jumping into its genes. The piggyBac itself is 2.5 kb long with 13 bp inverted terminal repeats. It has specificity for the base sequence TTAA (at which it inserts); the probability of this sequence occurring is (0.25) 4 or 0.4 percent in any stretch of DNA, where it can cause insertion mutations: disrupting and inactivating genes, or inappropriately activating genes. This transposon was later found to be active in a wide range of species, including the fruit fly Drosophila , the mosquito transmitting yellow fever A aegypti , the medfly Ceratitis capitata , and the original host, the cabbage looper. The piggyBac vector gave high frequencies of transpositions, much higher than other transposon vectors in use, such as the mariner and Hirmar. The piggyBac transposon is also active in human and mouse cells, and in the mouse germline; and a version with minimal terminal repeats exhibited greater transposition activity in human cells than another, well-characterised hyperactive Sleeping Beauty transposon system widely used for preclinical gene therapy studies.
Tomorrow ..A safer solution perhaps