Abstract:
Chickens serve as an excellent model organism for developmental biology, offering unique opportunities for precise spatiotemporal access to embryos within eggs. Optogenes are light-activated proteins that regulate gene expression, offering a non-invasive method to activate genes at specific locations and developmental stages, advancing developmental biology research. This study employed the Magnet-Cre optogenetic system to control gene expression in developing chicken embryos. Magnet-Cre consists of two light-sensitive protein domains that dimerize upon light activation, each attached to an inactive half of the Cre recombinase enzyme, which becomes active upon dimerization. We developed an all-in-one plasmid containing a green fluorescent protein marker, the Magnet-Cre system, and a light-activated red fluorescent protein gene. This plasmid was electroporated into the neural tube of Hamburger and Hamilton (H&H) stage 14 chicken embryos. Embryo samples were cleared using the CUBIC protocol and imaged with a light sheet microscope to analyze optogenetic activity via red-fluorescent cells. We established a pipeline for Magnet-Cre activation in chicken embryos, demonstrating that a single 3-min exposure to blue light following incubation at 28 °C was sufficient to trigger gene activity within the neural tube, with increased activity upon additional light exposure. Finally, we showed a spatiotemporal control of gene activity using a localized laser light induction. This research lays the groundwork for further advancements in avian developmental biology and poultry research, enabling spatiotemporal control of genes in both embryos and transgenic chickens.
