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

Autor: Maharjan M; Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America., Maeda RK; Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland., Karch F; Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland., Hart CM; Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2018 Oct 08; Vol. 13 (10), pp. e0205538. Date of Electronic Publication: 2018 Oct 08 (Print Publication: 2018).
DOI: 10.1371/journal.pone.0205538
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
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