An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases

Abstract

Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a C31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the C31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the C31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, highthroughput screening of large cDNA sets and regulatory elements.attP landing sites ͉ germ-line transformation ͉ site-specific integration A major goal in the present era of genomics is to identify and functionally characterize all genes relevant to a specific pathway or biological process. With its powerful repertoire of genetic tools, the multicellular model organism Drosophila melanogaster has played an eminent role in this endeavor (1). One method to identify relevant genes is to perform chemical mutagenesis screens of various kinds. Another very fruitful approach in Drosophila has been the use of P-element-mediated germ-line transformation (2, 3), especially when combined with tools such as the Gal4/UAS expression system (4) or when it is used for insertional mutagenesis (5, 6). One characteristic of P-elements is their random integration behavior. Although this ''randomness'' is advantageous for generating mutations and deletions, it is generally not ideal for transgene analysis. The random integration of P-elements necessitates considerable effort to map insertions. Genomic position effects complicate the analysis of transgenes and render precise structure/ function analyses nearly impossible. A further shortcoming of the P-element system is its relatively moderate transformation efficiency, a significant hurdle to any large-scale transgenesis effort.Strategies have been developed to circumvent the problem of randomness by targeted integration systems in Drosophila, which are generally based on the FLP and Cre recombinases (7-9, 32). Such techniques permit precise targeting to genomic landing sites but are still handicapped by transformation rates that are, at best, moderately higher than those achieved with the P-element system (8). Furthermore, especially for FL...

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

“…UAS‐SPOP transgenic flies were generated by injecting transgenes and targeting them to the 2R‐51D landing site using the PhiC31 system (Bischof et al , 2007). Bestgene Inc performed the injections.…”

Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes

“…Moreover, maximal inducibility in one tissue does not guarantee maximal inducibility in another tissue. Thus, the standard practice in Drosophila of generalizing transgene inducibility on the basis of expression in a single tissue-most commonly using the eye and more recently the wing disc 25 -is of limited use in assessing how well a transgene will be expressed in other tissues. Our results demonstrate instead that transgene activity must be empirically determined for each tissue.…”

“…The phiC31 integrase mediates recombination between the bacterial and phage attachment sites, attB and attP, and has been shown to efficiently integrate attB-containing plasmids into attP 'landing sites' that have been previously inserted in the genome 22 . To date, over 100 attP landing-site loci have been randomly integrated into the Drosophila genome [22][23][24][25] . Some of these landing sites have been modified to allow for recombinase-mediated cassette exchange 23 and the unambiguous detection of integrated transgenes 25 , whereas others have been shown to be amenable to the integration of large 100-kb DNA constructs 24 .…”

“…To date, over 100 attP landing-site loci have been randomly integrated into the Drosophila genome [22][23][24][25] . Some of these landing sites have been modified to allow for recombinase-mediated cassette exchange 23 and the unambiguous detection of integrated transgenes 25 , whereas others have been shown to be amenable to the integration of large 100-kb DNA constructs 24 . However, it remains unclear whether position effects at any of these attP sites permit optimal transgene expression.…”

p37δ is a new isoform of PI3K p110δ that increases cell proliferation and is overexpressed in tumors

“…PIK3CD_v2 were also cloned into the pUASTattB vector, kindly provided by Bischof et al (2007). All constructs were verified by BigDye Cycle Sequencing (Applied Biosystems).…”