Fly RMCE
phiC31-mediated RMCE
Transgene studies are often plagued by position
effects arising from
the chromosomal regions flanking insertion sites. Not only do
such position effects complicate the functional analysis of any single
transgene, they preclude straightforward comparisons of transgenes that
have inserted randomly into different genomic regions. To address these issues, we have developed a technique for the precise placement of transgenes into predetermined sites in the Drosophila genome. This method adapts the technology of recombinase-mediated cassette exchange (RMCE) to use the recombination machinery of the phiC31 integrase. Essentially, it allows researchers to exchange a target cassette located in the genome with a donor cassette carried on plasmid by inducing phiC31 integrase-mediated recombination on both sides of the aligned cassettes.
We have generated a number of Drosophila lines that each carry a target mini-white cassette flanked by attP sites. In order to use these lines for transformation, one must simply flank a gene of interest with inverted attB sites (which may be done by cloning the gene into the vector piB-GFP, or by other cloning strategies). A more detailed description of the technique is available in our manuscript.
*Note that the orientation of the attB sites is important in order to insert the sequence of interest rather than the plasmid backbone when using our target lines. Both attB and attP have a central TTG where the crossover takes place; in all vectors, we have oriented the TTG to read into the exchange cassette on the top strand from both directions (i.e. no matter which strand you read 5'-3', you should read "TTG-cassette-CAA").
Further Reading (including articles on RMCE using Cre or FLP):
Baer A, Bode J. 2001. Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted intergration of transgenes. Curr Opin Biotech 12: 473-80.
Groth AC, Fish M, Nusse R, Calos MP. 2004. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166: 1775-82.
Oberstein A, Pare A, Kaplan L, Small S. 2005. Site-specific transgenesis by Cre-mediated recombination in Drosophila. Nat Methods 2: 583-5.
Horn C, Handler AM. 2005. Site-specific genomic targeting in Drosophila. Proc Natl Acad Sci USA 102: 12483-8.