Multiplexed Retinoic Acid Signal Reporters for In Vivo and In Vitro Use
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
ABSTRACT Here we describe the optimisation of a modular, generally applicable and rapid method to make multimerised signaling‐molecule reporters for detecting different retinoic acid concentrations. The technical plasmid design allows for the easy exchange of reporters, promoters, signal‐responsive elements as well as for the multimerization of entire cassettes of these modular units. These constructs are ideal for pronuclear injection resulting in random integration into genomic DNA so avoiding the use of embryonic stem cells for generating transgenic lines or embryos. The use of insulator elements was shown to isolate reporter cassettes from integration‐specific influences and from adjacent cassettes. Such insulators were found to be beneficial in transient transfections of primary cells, where DNA would not have integrated, as seen by more accurate ligand concentration responsiveness. The choice of promoter is important as weak promoters in in vitro experiments did not function in transgenic embryos. Notably, the core binding site and surrounding nucleotides could be used to modulate retinoic acid responsiveness in vitro. However, in vivo, the construct not only reflected retinoic acid concentrations but also affected tissue specificity. Increasing the number of retinoic acid receptor binding sites from 4 to 12 copies increased in vitro RA sensitivity, but had a lesser effect on in vivo sensitivity. We observed robust expression in the primitive streak of presomitic embryo
Abstract
ABSTRACT Here we describe the optimisation of a modular, generally applicable and rapid method to make multimerised signaling‐molecule reporters for detecting different retinoic acid concentrations. The technical plasmid design allows for the easy exchange of reporters, promoters, signal‐responsive elements as well as for the multimerization of entire cassettes of these modular units. These constructs are ideal for pronuclear injection resulting in random integration into genomic DNA so avoiding the use of embryonic stem cells for generating transgenic lines or embryos. The use of insulator elements was shown to isolate reporter cassettes from integration‐specific influences and from adjacent cassettes. Such insulators were found to be beneficial in transient transfections of primary cells, where DNA would not have integrated, as seen by more accurate ligand concentration responsiveness. The choice of promoter is important as weak promoters in in vitro experiments did not function in transgenic embryos. Notably, the core binding site and surrounding nucleotides could be used to modulate retinoic acid responsiveness in vitro. However, in vivo, the construct not only reflected retinoic acid concentrations but also affected tissue specificity. Increasing the number of retinoic acid receptor binding sites from 4 to 12 copies increased in vitro RA sensitivity, but had a lesser effect on in vivo sensitivity. We observed robust expression in the primitive streak of presomitic embryos and in the somites, neural tube and presomitic mesoderm of 3–10‐somite embryos. Notably, use of only two copies of the binding site resulted in construct‐inherent variability in cell type specificity and RA concentration sensitivity. This was conclusively demonstrated using dual receptors, which inherently control for confounding integration or copy‐number dependent effects. In conclusion, the use of dual reporters with insulators is a useful approach for concurrently measuring concentration and context dependent responses to one signaling pathway and also lends itself for examining the interaction of two signaling pathways within one embryo.