Mosquito Transposon-Mediated Transgenesis

Abstract

Transposon-mediated transgenesis of mosquito vectors of disease pathogens followed the early success of transgenesis in the vinegar fly, Drosophila melanogaster The P transposable element used in Drosophila does not function canonically in mosquitoes, and repeatable, routine transgenesis in mosquitoes was not accomplished until new transposable elements were discovered and validated. A number of distinct transposons were subsequently identified that mediate the introduction of exogenous DNA in a stable and heritable manner in mosquito species, including members of the genera Aedes, Anopheles, and Culex The most versatile element, piggyBac, is functional in all of these mosquito genera, as well as in many other insects in diverse orders, and has been used extensively outside the class. Transposon-mediated transgenesis of recessive and dominant marker genes and reporter systems has been used to define functional fragments of gene control sequences, introduce exogenous DNA encoding products beneficial to medical interests, and act as "enhancer traps" to identify endogenous genes with specific expression characteristics.

FIGURE 1.

General organization and use of Class II transposable elements. (A) Schematic representations of a Class II transposable element and engineered helper and donor plasmids. Active Class II transposable elements have inverted terminal repeated (ITR) DNA sequences flanking a functional transposase gene complete with promoter and non-transcribed control DNA (Transposase). The helper plasmid (Helper) has the transposase open reading frame (ORF) under the control of a constitutive promoter (Con-pro). The donor plasmid (Donor) contains ITRs flanking genes of interest, a dominant marker gene along with control sequences to allow visible screening of transgenic animals, and in some cases sites for site-specific recombination (SSR). Both donor and helper DNA sequences are cloned into bacterial plasmids (thin lines) that allow production and purification for use in embryo microinjections or other DNA-introduction technologies. (Table) Examples of promoter sequences and marker/reporter genes along with early-or first-use citations in mosquitoes. (B) Examples of transposon-mediated transgenic mixed-instar Anopheles stephensi mosquito larvae marked with 3xP3-fluorescence genes. All larvae show transgene-mediated fluorescence in their eyes (E). Some larvae exhibit additional transgene-specific fluorescence in the segmented nervous tissue (vertical brackets), and anal gills (AG). The food bolus in the gut can produce background (BK) fluorescence. Horizontal bars, ∼1 mm. (EGFP) Enhanced green fluorescent protein, (CFP) cyan fluorescent protein, (DsRed) Discosoma species red fluorescent protein.