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

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.