The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA “in cubo”, followed by the incubation of the electroporated embryo and microscopy of the electroporated region with the embryo in the cube. The egg-in-cube culture system in combination with the “in cubo” electroporation and dynamic imaging capabilities described here will enable researchers to investigate several fundamental questions in early embryogenesis with the avian (quail) embryo on its native yolk.
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