Single copy transgenes in the chicken genome have greater stability, uniformity of expression, and hence greater utility than electroporated constructs for applications such as cell lineage tracing. Embryos carrying a single copy GFP transgene, from the ubiquitous GFP transgenic chicken line Roslin Green, have been used in numerous developmental biology investigations (McGrew et al., 2008).The Notch signalling pathway is a key regulator of neurogenesis in the vertebrate neural tube. We are developing a fluorescent reporter transgenic chicken line that should allow Notch signalling through the Notch-responsive gene Hes5-1 to be tracked in all tissues of the embryo throughout development. A Venus fluorescent reporter driven by the Hes5-1 promoter with destabilising elements was developed in 2011 by Vilas-Boas and Storey (Vilas-Boas et al., 2011) as a reporter of Notch signalling in the developing chicken neural tube. We cloned this reporter construct into a lentiviral vector and, after lentiviral packaging, the pseudovirus containing the transgene was injected into HH stage X chicken embryos which were incubated to hatch. One surviving chimeric transgenic cockerel was identified and bred to produce offspring, including two transgenic cockerels each carrying a single copy of the transgene. These G1 cockerels are now being bred to produce G2 transgenic embryos for analysis. We describe progress in validation of these transgenic embryos as reporters of Notch signalling in the developing neural tube.Other fluorescent reporter transgenic chicken lines in progress at various stages of production will be described and the principles behind their development explained. These include a transgenic chicken line designed to ubiquitously express the red fluorescent protein tdTomato. In addition to lentiviral transgenesis, transposon-based methods for generating transgenic chicken lines are being explored, using a piggyBac transposon vector to introduce transgenes into primordial germ cells. Constructs developed include a piggyBac-CAG-Brainbow2.1 transposon. Such fluorescent reporter transposons could also prove useful for long-term lineage tracking in ovo, by co-electroporation with transposase.
|Publication status||Published - 5 Mar 2014|
|Event||Avian Model Systems - Cold Spring Harbor, United States|
Duration: 5 Mar 2014 → 8 Mar 2014
|Conference||Avian Model Systems|
|City||Cold Spring Harbor|
|Period||5/03/14 → 8/03/14|