Other Approaches To Genetic Engineering In Insects

FLP/FRT Recombinase in Nondrosophilid Insects

The FLP/FRT recombinase system of the yeast Saccharomyces cerevisiae can also function correctly in at least one nondrosophilid species. Plasmid-based excision and integration assays showed that the FLP recombinase enzymes could recognize and recombine FRT sites in the soma of developing Ae. aegypti embryos. Excision at the FRT sites was high—60% of plasmids examined had undergone an excision event mediated by FLP recombinase. Integration, as measured by the formation of heterodimeric plasmids arising from the recombination between two plasmids each containing an FRT site, occurred at a low, but statistically significant, frequency. The ability of the FLP/FRT recombinase system to function correctly in Drosophila and Aedes suggests that it should function across a range of insect species. It cannot, however, be used to directly genetically transform an insect species because to achieve this, FRT sites must first be introduced into the target genome by some other means, such as transposable elements. When combined with transposable element technology, this yeast recombination system should allow investigators to undertake precise manipulations of both introduced and host DNA. This ability will be of particular importance if DNA sequences necessary for the movement of transposable elements need to inactivated, (e.g., for regulatory reasons) following initial integration of the element into the target genome.

RNA-Mediated Interference (RNAi) in Insects

RNA-mediated interference (RNAi) refers to the targeted disruption of gene expression arising from the introduction of double-stranded RNA (dsRNA) into the cell. This disruption is targeted only to RNA molecules homologous to the invading dsRNA. It was initially characterized in plants and in the nematode C. elegans but is now thought to be a general phenomenon of eukaryotic cells that enables them to overcome invasions of RNA viruses. The mechanism by which RNAi works is unknown. It does not work through a simple titration of nascent or messenger RNA as would occur for an antisense RNA-based mechanism because the RNAi response can be elicited by far fewer dsRNA molecules per cell than, target RNA molecules. A catalytic mechanism in which the presence of dsRNA induces the destruction of homologous cellular RNAs has been recently proposed. RNAi technology has been harnessed to allow the targeted inactivation of specific genes and will prove to be a valuable component of genomics projects in those species in which nucleic acids can be introduced into cells. In its original experimental design, the effects of RNAi were not inherited. RNAi technology has recently been combined with P transposable element technology in D. melanogaster to produce heritable RNAi-mediated gene inactivation. Thus it is possible to examine the function of genes expressed in later stages of development of this insect and also the generation of genetically stable mutant lines in which production of the dsRNA can be induced or terminated based on the promoter used to drive expression of the extended hairpin loop RNA. RNAi technology should be extendable into other insect species in which transformation systems exist, and its exploitation in insects such as mosquitoes will enable the effects of the selective inactivation of specific genes to be quickly determined. This will represent a significant advance over traditional methods of creating and isolating mutants in these insect species that have not traditionally been amenable to genetic analyses.

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