High frequencies of Minos transposon mobilization are obtained in insects by using in vitro synthesized mRNA as a source of transposase.
One of the most frequently encountered problems in transposon-mediated transgenesis is low transformation frequency, often resulting from difficulty in expressing from injected plasmid DNA constructs adequate levels of transposase in embryos. Capped RNA corresponding to the spliced transcript of the Minos transposable element has been synthesized in vitro and shown to be an effective source of transposase protein for Minos transposon mobilization. Transposase produced by this mRNA is shown to catalyze excision of a Minos transposon from plasmid DNA in Medfly embryos. When injected into Drosophila or Medfly embryos, transposase mRNA leads to a several-fold increase in transformation efficiencies compared with injected plasmids expressing transposase. Also, frequent mobilization of a Minos transposon from the X chromosome into autosomes was demonstrated after injections of Minos transposase mRNA into pre-blastoderm Drosophila embryos. The high rates of transposition achieved with transposase mRNA suggest that this is a powerful system for genetic applications in Drosophila and other insects.
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, PO Box 1527, Vassilika Vouton, Heraklion-Crete 71110, Greece.
Maria G
MG
Kapetanaki
Thanasis G
TG
Loukeris
Ioannis
I
Livadaras
Charalambos
C
Savakis
D000818:Animals; D030801:Animals, Genetically Modified; D004251:DNA Transposable Elements; D004175:Diptera / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics; D018095:Germ-Line Mutation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000378:metabolism; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D019895:Transposases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D012333:RNA, Messenger; D019895:Transposases