Department of

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

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.

Stem cells organize themselves into pseudo-embryos

Scientists have developed murine artificial pseudo- embryos capable of forming the three major axes of the body. An innovative tool to potentially replace mammalian embryos in many experimental conditions.

Structural molecular biology of RNA processing machines

19.10.2018 14:00, 3079 (Sciences III)

Yunsun Nam (University of Texas Southwestern, USA).
hosted by: Ramesh Pillai.