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

PLoS One. 2018 Oct 8;13(10):e0205538. doi: 10.1371/journal.pone.0205538. eCollection 2018.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Animals
  • Animals, Genetically Modified
  • Attachment Sites, Microbiological*
  • Bacteriophages
  • Binding Sites
  • Chromosomal Position Effects*
  • Compound Eye, Arthropod / metabolism
  • DNA Transposable Elements
  • Drosophila / genetics*
  • Drosophila / metabolism
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Enzymes
  • Eye Proteins / genetics
  • Eye Proteins / metabolism
  • Female
  • Genetic Engineering / methods*
  • Genome

Substances

  • ATP-Binding Cassette Transporters
  • DNA Transposable Elements
  • Drosophila Proteins
  • Enzymes
  • Eye Proteins
  • w protein, Drosophila
  • y protein, Drosophila

Grant support

This work was supported by the National Science Foundation [1244100 to CMH]; the State of Geneva [FK and RM]; the Swiss National Fund [FK and RM]; and Donation Claraz [FK and RM]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.