Hello all,
I work in a university laboratory where we study the mechanisms behind sex change in clownfish, our latest project involves making transgenic clownfish which has stumped us for over a year. We have injected hundreds of thousands of eggs and have produced a single transgenic clownfish. Our biggest problems involve keeping eggs alive and hatching them.
To make the transgenic fish, we take eggs that have been laid within an hour and use a 5uM needle to inject about 0.5nL of transgenic reagent into the egg. This presents a variety of problems. First, the parents somehow know that their eggs have been compromised and eat them, so we have to artificially incubate them. Artificial incubation has way worse survival than the fathers care for unclear reasons, but we have gotten to the point where we get a fair few eggs to hatch out semi regularly, where our main problem lies which is what I hope you guys can help with.
For whatever reason, these transgenic embryos (which we have confirmed prior to hatching) have a very hard time hatching out. One of our colleagues did a similar project and was incapable of getting them to hatch, he had to manually remove the embryo from the egg to get any surviving larvae which is a very difficult process due to the size of the eggs. We have tried varying a variety of parameters to see if we can make them hatch like aeration, rotifer and nanochloropsis concentration, and my latest experiment involves using a lamp to simulate moonlight to hopefully trigger them to hatch. The thing is we have done these exact same practices with non injected eggs and gotten fantastic survival, which makes me think something about being injected might scar the egg shell or something that makes it harder for the embryo to break out. Usually, in my experience, if they havent hatched by 8 days old (where the day they were laid was day 1), they dont hatch at all.
Do you guys have any tips or tricks to stimulate them to hatch? Any advice in general would be greatly appreciated>
I work in a university laboratory where we study the mechanisms behind sex change in clownfish, our latest project involves making transgenic clownfish which has stumped us for over a year. We have injected hundreds of thousands of eggs and have produced a single transgenic clownfish. Our biggest problems involve keeping eggs alive and hatching them.
To make the transgenic fish, we take eggs that have been laid within an hour and use a 5uM needle to inject about 0.5nL of transgenic reagent into the egg. This presents a variety of problems. First, the parents somehow know that their eggs have been compromised and eat them, so we have to artificially incubate them. Artificial incubation has way worse survival than the fathers care for unclear reasons, but we have gotten to the point where we get a fair few eggs to hatch out semi regularly, where our main problem lies which is what I hope you guys can help with.
For whatever reason, these transgenic embryos (which we have confirmed prior to hatching) have a very hard time hatching out. One of our colleagues did a similar project and was incapable of getting them to hatch, he had to manually remove the embryo from the egg to get any surviving larvae which is a very difficult process due to the size of the eggs. We have tried varying a variety of parameters to see if we can make them hatch like aeration, rotifer and nanochloropsis concentration, and my latest experiment involves using a lamp to simulate moonlight to hopefully trigger them to hatch. The thing is we have done these exact same practices with non injected eggs and gotten fantastic survival, which makes me think something about being injected might scar the egg shell or something that makes it harder for the embryo to break out. Usually, in my experience, if they havent hatched by 8 days old (where the day they were laid was day 1), they dont hatch at all.
Do you guys have any tips or tricks to stimulate them to hatch? Any advice in general would be greatly appreciated>
