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ISIS Report - March 15, 2001
Terminator Insects - A Primer
piggyBac a name to remember
The first genetically modified (GM) insects are due for release in
the United States this summer. Prof. Joe Cummins takes us through the
basic genetics and the hazards involved.
The first GM insects to be released are modified with a marker to allow
them to be easily traced. Later, female killing traits will be introduced in GM
insects in the hopes that insect pest can be eradicated by mating with GM
insects.
The GM insects to be released are modified using mobile genetic elements
called transposons. Such mobile genetic elements are being promoted as a means
of controlling weeds as well as insect pests. However, the dangers of using
mobile genetic elements to eradicate pests should be recognized and discussed,
and not left to the fatuous embellishments of those profiting
from the
technology.
The piggyBac transposon is used in genetic engineering of pest
insects because it can be transferred to a number of insect pests from moths
and flies to mosquitoes. Gene transfer in insects began in the early 1980s with
the P transposon of the fruit fly. That transposon was first recognized as the
cause of a phenomenon called hybrid dysgenesis. In crosses between
species of the fruit fly certain crosses led to severe genetic damage in off
springs, there were high levels of gene mutations and chromosome rearrangement
leading to weak and infertile individuals.
The cause of hybrid dysgenesis was traced to the release and spread of
the P transposon among
the chromosomes of the affected individual. The P
transposon proved valuable in gene transfer among Drosophila species but was
not active in other insects.
The piggyBac transposon was discovered in cell cultures of the
moth Trichopulsia, also known as the cabbage looper. The looper cells
caused extensive mutation in an insect Baculovirus (Fraser et al 1985).
The transposon is being used extensively as it acts as a gene transfer vector
for a number of unrelated insects. Transposons are related to viruses but lack
the ability to be packaged in a virion (virus capsule).
The piggyBac transposon is flanked by short inverted repeats that
initiate non-replicative insertion into the insect chromosome, mediated by an
enzyme called transposase that targets the sequence TTAA on the chromosome.
When the transposon inserts into a gene sequence, it may cause a mutation, or
the transposon may act as a recombination site leading to duplication and
deletion of sequences in the chromosomes.
The transposon is usually multiplied by being inserted in a bacterial
plasmid. The transposon bearing plasmids are injected into early insect
embryos, where they may insert into the genome of germ cells and become
maintained in a lineage arising from the germ cells. The plasmids injected into
an embryo include the transposon carrying the gene(s) to be transferred and
lacking a transposase, to ‘disable’ the transposon and prevent
it from moving by itself.
The transposase function is provided by a helper
plasmid that has a transposon with the transposase gene, but is otherwise also
disabled because it has one terminal short repeat missing,
so it cannot
insert into the chromosome.
The piggyBac transposon has been used, for example. to transform fruit
fly, yellow fever mosquito and cabbage looper (Lobo et al 1999); the silkworm
has been transformed using piggyBac (Toshiki et al 2000) as has pink
bollworm, a cotton pest (Peloquin et al 1999).
Field release of genetically modified pink bollworm
The United States Department of Agriculture (USDA) APHIS approved field
release of a genetically modified (GM) insect pest, the pink bollworm. Some
2,350 adult insects will be released over the year beginning July 15 2001 and
ending July 14 2002 in a small cotton field (3 acres) located .5 miles from the
Plant Protection Center Rearing facility, Phoenix, AZ, USA. The field test
application is available on line:
http://www.aphis.usda.gov/biotech/arthropod/permits/0102901r/0102901r.html
The application was submitted 17 January 2001. The GM insects have been
modified with a gene for the green fluorescent protein (GFP) from a jellyfish.
The GFP is regulated from the heat shock promoter (hsp promoter) of the fruit
fly. The bollworm was modified using the piggyBac transposon that
inserts at the sequence, TTAA in the chromosome ( see above). The genetic
modification includes the transposon short terminal repeats but lacks the
trasposase gene needed to mobilize the chromosomal insert. It is presumed that
the pink bollworm lacks the competent transposon and will not encounter viruses
harboring the piggyBac transposon or any other transposon that can
mobilize the insert in the host chromosome. The release experiment is believed
to be a precursor to using the GFP marker in evaluating the use of irradiated
males to control the bollworm pest and to lead to development of female killing
genetic systems capable of eradicating the bollworm pest.
The proposal for the field test is based on the belief of the
researchers that the piggyBac gene inserts would be stable. However, no
direct evidence of stability under laboratory conditions was provided.
Furthermore, piggyBac may be carried by insect baculovirus and such virus
certainly produce transposase that can mobilize the gene-bearing transposon.
Virus can act as a vector for rapid spread of the modified transposon to a
variety of insects.
The proposal argue that no human health concerns are involved in the
field trail. It argues "Lepidoptera, in general, do not pose a threat to human
health and welfare and should remain a guiding principle in deciding on human
risks related to their genetic manipulation." Even
though the principle
may have some merit it is clear that the piggyBac transposon was also used for
gene transfer in the human pathogen related yellow fever mosquito.
Even more significantly, the insect Baculovirus is known to carry
piggyBac transposon and that insect virus is used for human gene therapy. The
spread of piggyBac to produce green neighbors
that fluoresce may be
greeted with hilarity by USDA. However, the inserted transposon efficiently
creates mutations in genes such as recessive oncogenes associated with cancer .
The proposed field trials should be suspended until adequate laboratory
experiments are conducted on transposon gene stability and on both viral and
proviral helper functions that can mobilize disabled transposons carrying
transgenes.
In a following essay I will discuss the construction and the use of
female killing genes in GM insects to eradicate insect pests.
References
- Fraser,M,Brusca,J,Smith,G and Summers,M "Transposon mediated
mutagenisis of a baculovirus" 1985 Virology 145,356-61
- Lobo,N,Li,X and Fraser,M "Transposition of the pigyBac element in
embryos of Drosophila melangaster, Aedes aegypti and Trichoplusia ni"
1999 Mol Gen Genet 261,803-10
- Peloquin,J,Thibault,S,Staten,R and Miller,T "Germline transformation
of pink bollworm (Lepidoptera: Gelechidae) mediated by the piggyBac
transposable element" 2000 Insect Molecular Biology 9,323-33
- Tamura,T,Thibert,C,Corrine,R,Kanda,T, Abraham,E, Kamba,M, Komoto,N,
Thomas,J, Mauchamp,B, Chavency,G, Sirk,P, Fraser,M, Pruddhomme,J and Couble,P.
Germline transformation of the silkworm Bombyx mori L using a piggBac
transposon-derived vector" 2000 Nature Biotech 18,81-4
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