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An engineered phiC31 “Disintegrase” able to make an  attP  site in  Drosophila  out of an  attR - attL  pair is described. This was used to generate  attP  sites at genomic locations where a mini- white  (mini- w ) transgene was subject to chromosomal position effects (CPE). The first step was random genomic integration of a P-element-based transposon with an insulated mini- w  transgene. We then removed the upstream insulator using FLP recombinase to detect CPE. Next mini- w  and the downstream insulator were “dis-integrated” leaving behind an  attP  site. The location is marked by a  yellow   +   transgene that is flanked by  loxP  sites, so it can also be removed. Using this system, we generated 10 new  attP  landing platforms. Three of these showing strong activating CPE were selected for further analysis. We show that the  attP  sites are functional by integrating in plasmids with  attB  sites. The CPE is recapitulated and can be blocked by insulators. We show that a dimerized 215 bp fragment of the 500 bp BEAF-dependent scs’ insulator containing a high affinity BEAF binding site blocks the CPE, while a monomer of the sequence is less effective. This indicates that two BEAF binding sites make a stronger insulator than a single site. This system could be useful for generating  attP  sites at prescreened sites for other purposes, such as studying CPE in embryos or other tissues or for use with “trapped” enhancers of interest.

Using a phiC31 “Disintegrase” to make new attP sites in the Drosophila genome at locations showing chromosomal position effects