Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti

Derivatives of the mariner transposable element, Mos1, from Drosophila maur itiana, can integrate into the germ-line of the yellow fever mosquito, Aede s aegypti. Previously, the transposase required to mobilize Mos1 was provid ed in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. a egypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/ kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of t he wild-type allele of the D. melanogaster cinnabar gene. Transformed mosqu itoes were recognized by partial restoration of eye color in the G(1) anima ls and confirmed by Southern analyses of genomic DNA, At Mos1 transposase c oncentrations approaching 100 nM, the rate of germ-line transformants arisi ng from independent insertions in G(o) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recover y of total G(1) transformants was increased 7.5-fold over the frequency see n with co-injected helper plasmid. Southern blot analyses and gene amplific ation experiments confirmed the integration of the transposons into the mos quito genome, although not all integrations were of the expected cut-and-pa ste type transposition. The increased frequency of germ-line integrations o btained with purified transposase will facilitate the generation of Mos1 tr ansgenic mosquitoes and the application of transgenic approaches to the bio logy of this important vector of multiple pathogens. (C) 2000 Elsevier Scie nce Ltd. All rights reserved.