Fig. 1. Prediction of Foxn4 cis-regulatory elements and experimental design for functional verification.(A) Comparative sequence analysis between mouse.

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Fig. 1. Prediction of Foxn4 cis-regulatory elements and experimental design for functional verification.(A) Comparative sequence analysis between mouse and 9 other vertebrate Foxn4 loci revealed 4 evolutionarily conserved regions (CR). Prediction of Foxn4 cis-regulatory elements and experimental design for functional verification.(A) Comparative sequence analysis between mouse and 9 other vertebrate Foxn4 loci revealed 4 evolutionarily conserved regions (CR). For simplicity, only human, mouse, and chicken alignment is shown here. Blue peaks represent Foxn4 exons while pink peaks represent conserved non-coding sequence. (B) Design of plasmid reporter constructs for experimental construct, and various control constructs, i.e. the negative control, transfection control, and positive control. The experimental construct contains an enhancer candidate upstream of a minimal β-globin promoter and a reporter GFP. Negative control constructs contain the minimal β-globin promoter and the reporter GFP without an inserted sequence or with a random sequence of comparable size. The transfection control contains a strong ubiquitous CAG promoter (chicken β-actin promoter with CMV enhancer), which is in place of the β-globin minimal promoter. The positive control contains a known enhancer, e.g. the RER enhancer (Nie et al., 1996) for photoreceptors, to ensure GFP is expressed in a cell-type specific manner in the presence of a functional enhancer and the β-globin minimal promoter. (C) A mixture of plasmid DNA constructs including the experimental constructs and transfection control, CAG-DsRed was injected and electroporated into the chick retina at embryonic day 4 (E4) to transfect the retinal progenitor cells. Mohammed M. Islam et al. Biology Open 2013;2:1125-1136 © 2013. Published by The Company of Biologists Ltd