Stable, Germ-Line Transformation ofCulex quinquefasciatus(Diptera: Culicidae)

Abstract

A Hermes-based transposable element transformation system incorporating an enhanced green fluorescent protein (EGFP) marker was used to produce two transgenic lines of Culex quinquefasciatus (Say). The transformation frequency was approximately 12% and transformation of Culex was shown to be dependent on the presence of Hermes transposase. Injected Culex embryos were treated with four different heat shock regimes, two of which produced transformed individuals. These individuals were mated with wild-type mosquitoes and produced offspring which expressed the dominant EGFP gene in Mendelian ratios predicted for the stable integration of a gene at a single locus. The two transformed lines displayed distinct patterns of phenotypic expression, the expression of which has remained stable after fifteen generations. In these transgenic lines both the Hermes element and flanking plasmid DNA integrated into the Culex genome, as has been previously seen in Hermes-mediated transgenic strains of Aedes aegypti (L.). The high frequency of Culex transformation together with the dependence on the presence of Hermes transposase suggests that, as for Ae. aegypti, this mode of transposition into the germ-line genome occurs by an alternate mechanisms to the cut and paste type of transposition seen for this element in other insect species and in the somatic nuclei of mosquitoes. This is the first report of the genetic transformation of a species in the genus Culex and demonstrates that this medically important mosquito species can now, along with several other Culicine and Anopheline mosquito species, be genetically manipulated.

Gene vector and transposable element behavior in mosquitoes

“…Canonical cut-and-paste type transposition events, however, have never been observed in the germ lines of mosquitoes transformed using Hermes vectors. For both A. aegypti and Culex quinquefasciatus, germ-line integrations have involved not only all of those sequences delimited by the terminal inverted repeats of Hermes but also sequences flanking the element on the vector-containing plasmid (Allen et al, 2001;Jasinskiene et al, 1998Jasinskiene et al, , 2000Kokoza et al, 2000;Moreira et al, 2000;Pinkerton et al, 2000).…”

piggyBac-mediated Germline Transformation of the Malaria Mosquito Anopheles stephensi Using the Red Fluorescent Protein dsRED as a Selectable Marker

“…Although EGFP has proven to be an invaluable visible marker for identifying transformed individuals in different insect species (1,(5)(6)(7), the availability of only this selectable marker limits the range of applications of a gene transfer technology in malaria vectors. New molecular tools are now needed to exploit fully the potential of germline transformation for Anopheles mosquitoes, with the aim of unraveling the interactions between host molecules and Plasmodium parasites, as well as performing functional studies such as transposon tagging and enhancer trapping.…”

Genomic resources for invertebrate vectors of human pathogens, and the role of VectorBase

“…The same transposon was employed by Adelman (2004) to transform somatic cells in A. aegypti. The Hermes and Mariner transposons were successfully used to transform respectively Culex pipiens quinquefasciatus germ line (Allen et al, 2001) and A. aegypti somatic cells (Coates et al, 1998). In addition, the availability of the genome sequence facilitates the development of high-throughput genome wide technologies such as high density expression micro-array, genome tiling array, or chipchip methodology.…”