title: Generating customized transgene landing sites and multi-transgene arrays in Drosophila using phiC31 integrase.
abstract: Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems.
authors: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147 knappj@janelia.hhmi.org simpsonj@janelia.hhmi.org.
authors: Jon-Michael
authors: JM
authors: Knapp
authors: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147.
authors: Phuong
authors: P
authors: Chung
authors: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147 knappj@janelia.hhmi.org simpsonj@janelia.hhmi.org.
authors: Julie H
authors: JH
authors: Simpson
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
chemical_list: D029721:Drosophila Proteins; D019426:Integrases
keywords: integrase; landing site; phiC31; shuffle; transgene array