title: Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti.
abstract: Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided 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. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA. At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(0) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, CA 92697-3900, Irvine, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Coates
authors.forename: N
authors.initials: N
authors.lastname: Jasinskiene
authors.forename: D
authors.initials: D
authors.lastname: Morgan
authors.forename: L R
authors.initials: LR
authors.lastname: Tosi
authors.forename: S M
authors.initials: SM
authors.lastname: Beverley
authors.forename: A A
authors.initials: AA
authors.lastname: James
mesh_terms: D000330:Aedes / Q000201:enzymology / Q000502:physiology*; D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D015139:Blotting, Southern; D004247:DNA; D004251:DNA Transposable Elements / Q000235:genetics*; D004330:Drosophila / Q000201:enzymology / Q000235:genetics*; D005784:Gene Amplification; D005854:Germ Cells; D019895:Transposases / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D004247:DNA; D019895:Transposases