Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element

Abstract

Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.

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

“…An EGFP-loaded piggyBac transposon has also been recently validated as DNA delivery vector in Anopheles gambiae mosquitoes, although at frequencies much lower than those obtained in this study (6). However, when comparing piggyBac to Minos, the fact that the latter element generally creates single integrations may represent an advantage in terms of studying the effects of introducing foreign DNA into the genome of heterologous organisms (1).…”

“…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.…”

Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

“…stephensi 21 and the discovery that the piggyBac transposable element could mediate germ-line transformation in many different insect species, Grossman et al 22 succeeded in producing An. gambiae transgenic mosquitoes.…”

“…stephensi and Anopheles albimanus, transformation efficiency was much lower in An. gambiae and more technically challenging 22,23 . Since then, several modifications to the protocol have improved the efficiency considerably, as outlined in Lobo et al 24 .…”

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

“…Handler (2002) describes how the piggyBac transposon allows germ-line transformation of insects, including the yellow fever and malaria vectors Aedes aegypti , A. gambiae (Grossman et al, 2001), Anopheles fluviatilis (Rodrigues et al, 2006), Anopheles stephensi (Nolan et al, 2002) and Anopheles albimanus (Perera et al, 2002). The same transposon was employed by Adelman (2004) to transform somatic cells in A. aegypti.…”