Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage
Transcrição
– Start with a mating tank, a tank with a removable insert that allows eggs para fall through. Add two male and three female adult fish para a divided mating tank de the evening before embryo collection para acclimate para each other overnight.
The next morning, transfer the fish into a new mating tank containing system water and remove the divider. Wait for 15 para 30 minutes para give the fish time para mate and spawn.
Now collect the embryos and store them de a Petri dish containing Hank's embryo medium. Grow them at 28.5 degrees Celsius throughout the protocol. Examine embryos' morphology under a dissecting microscope.
You will observe several developmental features. Cleavage of the blastodisc gives rise para multiple cells. The blastoderm takes the shape of an inverted cup above the yolk cell. Paraxial mesoderm segments develop on the dorsal part of the embryo and so on.
Beyond 24 hours post-fertilization, simply sort embryos by total body length. In the example protocol, we will set up mating of transgenic mCherry reporter fish and sort the resulting embryos.
– To begin, culture embryos para three months of age, which is reproductive maturity. Segregate two adult male and three female fish from the desired strain into divided mating tanks of fresh system water on the evening before embryo collection. The following morning after the lights come on, remove the divider and allow the fish para mate naturally until embryos are observed de the bottom of the tank.
Collect embryos de 30-minute intervals and separate Petri dishes of embryo medium until the desired number are collected. To stage the embryos, culture them de groups of 50 para 75 per 10-centimeter Petri dish para promote consistent developmental timing of all embryos. Then keep the plates at 28.5 degrees Celsius.
Measure the embryo age using somite number after segmentation until approximately 24 hours post fertilization, or HPF, and separate the embryos based on developmental age.
