Enhancer Identification using ZED Reporter

Candidate enhancer sequences of different alleles with closely flanking sequences were constructed into ZED Vector (Bessa et al. 2009 (link)) using Gateway Recombination Cloning Technology (Thermo Fisher Scientific). T7-Transposase (Khattak et al. 2014 (link)) (Addgene) was transcribed using mMESSAGE mMACHINE T7 kit (Life Technologies), according to the manufacturer’s instructions. A final concentration of 40 ng/µl ZED constructs, 50 ng/µl transposase mRNA, and 0.05% phenol red were coinjected into one-cell stage embryos. The embryos were imaged for GFP and internal control red fluorescent protein (RFP) expression at different time points using a Leica MZFLIII microscope. The elements were considered as candidate enhancers if there were more than 20% of injected embryos showing consistent expression pattern of GFP at the presence of RFP (Bessa et al. 2009 (link); Sharma et al. 2015 (link)).