Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid.
We describe a microinjection-based phiC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the phiC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.
Fish
Matthew P
MP
Department of Developmental Biology and Howard Hughes Medical Institute, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5120, USA. mattfish@stanford.edu
Groth
Amy C
AC
Calos
Michele P
MP
Nusse
Roel
R
D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D004330:Drosophila / Q000196:embryology / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D019426:Integrases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013302:Streptomyces / Q000821:virology; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics
D012333:RNA, Messenger; D014764:Viral Proteins; D019426:Integrases