Help me make transgenic clownfish

[Sharkbait19]

Do you have any information on the process used to make glofish that may point you in the right direction?

 

[dedragon]

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

How are you artificially incubating the eggs? Does it change the night of hatch?

 

[Sirduckington]

I missed this

"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."

in the original post.

I dont understand whats the question, if your specific manipulation is the reason for the low hatch rate, its you who knows exactly what you changed, how could others point out issues in what you ve done specifically.

Not sure about goldfish but in A. burtoni cichlids they did the exact same thing as us and theirs were able to hatch. I'm not sure if this is because A. burtoni embryos are stronger or the egg is thinner or what but from what I understand they didn't need to do anything special to get them to hatch.

Do you have any information on the process used to make glofish that may point you in the right direction?

 

[Sirduckington]

How are you artificially incubating the eggs? Does it change the night of hatch?

Yeah so we've been comparing two artificial incubation methods but there doesnt seem to be any significant difference. One is a tank with UV sterilization and the other is a tank with methylene blue. In these tanks eggs glued to a tile with an airstone vigorously aerating them until they are 7 days old. Then, they are moved to a separate tank with rotifers and nanochloropsis with all sides blacked out to prevent lateral light. Aeration is also decreased to just gently knock around the eggs for hatching because too vigorous aeration kills the larva once they've hatched, they're very sensitive to flow.

 

[Sirduckington]

I missed this

"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."

in the original post.

I dont understand whats the question, if your specific manipulation is the reason for the low hatch rate, its you who knows exactly what you changed, how could others point out issues in what you ve done specifically.

I'm asking if there is any way to improve hatching rates/ stimulate hatching as these eggs need all the help they can get and there isn't much I can do about the injections

 

[Jmp998]

If they are dying that early and at that high of a rate, it is probably not even related to the transgene. More likely a component of the buffer solution you are injecting, or even just the disruption of the cell membrane/underlying cytoskeleton during transfer, is interfering with normal development. You need to take a step back, try step by step to isolate the problem. For example poke but not inject, try injecting plain saline, etc to see if you can isolate which step of the manipulation is the problem. It may be something as simple as eliminating a toxic or interfering component in the injection buffer.

 

[c_healy]

Yeah so we've been comparing two artificial incubation methods but there doesnt seem to be any significant difference. One is a tank with UV sterilization and the other is a tank with methylene blue. In these tanks eggs glued to a tile with an airstone vigorously aerating them until they are 7 days old. Then, they are moved to a separate tank with rotifers and nanochloropsis with all sides blacked out to prevent lateral light. Aeration is also decreased to just gently knock around the eggs for hatching because too vigorous aeration kills the larva once they've hatched, they're very sensitive to flow.

Have you tried turning down the aeration earlier?

 

[Sirduckington]

If they are dying that early and at that high of a rate, it is probably not even related to the transgene. More likely a component of the buffer solution you are injecting, or even just the disruption of the cell membrane/underlying cytoskeleton during transfer, is interfering with normal development. You need to take a step back, try step by step to isolate the problem. For example poke but not inject, try injecting plain saline, etc to see if you can isolate which step of the manipulation is the problem. It may be something as simple as eliminating a toxic or interfering component in the injection buffer.

I think this is exactly right I've been wanting to do sham injections just never had the time but I think this is a really good idea. I have also experimented with not removing anything but just altering the total size of the injection and seen success which makes me think either the osmotic balance was disrupted or just too many mutations were occurring at the concentration I started at. Regardless, I think next chance I get I'll do a sham injection or maybe not even inject anything

 

[Sirduckington]

Have you tried turning down the aeration earlier?

I haven't, do you think high aeration during incubation could be harming them? My reasoning was it keeps the eggs clear of debris and microorganisms as well as keeps them oxygenated.

 

[dstockwell]

Mutant fish, why not just AI fish

 

[C4ctus99]

This may have been the most entertaining thing I read all day… I also originally thought the OP meant “transgender” and was over here thinking “they’re hermaphrodite…”

Good luck on your experiments! Sounds interesting and like there is definitely a point of failure somewhere in the injection. I haven’t tried injecting fish eggs with new genes yet though so I can’t help you too much

 

[Atherial]

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'm very curious as to why you are using clownfish as your model organism. This technique is very simple in zebrafish and well studied. Also why are you doing injections with random insertion instead of CRISPR?

As for the rest, are you pulling your own needles or buying them? How much are you injecting?

 

[Sirduckington]

I'm very curious as to why you are using clownfish as your model organism. This technique is very simple in zebrafish and well studied. Also why are you doing injections with random insertion instead of CRISPR?

As for the rest, are you pulling your own needles or buying them? How much are you injecting?

We are using Tol2 instead of crispr because from what I understand crispr is much better for knocking out genes whereas Tol2 is better for inserting genes with the caveat that you cant control where (I still think crispr would be a good idea but I'm not the PI lol) . We are pulling our own needles and found that egg survival gets worse the larger the needle is (seems obvious in hindsight). We started at 1nL but I've found that that's too much so now we are between 0.5 to 0.25 nL

 

[Sirduckington]

I'm very curious as to why you are using clownfish as your model organism. This technique is very simple in zebrafish and well studied. Also why are you doing injections with random insertion instead of CRISPR?

As for the rest, are you pulling your own needles or buying them? How much are you injecting?

Also we are using clownfish because we want to manipulate the hypothalamus to see how that affects the sex changing process so we want to insert designer receptors so we can artifically activate or inactivate certain cells and see how that affects things like E2 or 11KT production among other things.

 

[Atherial]

Also we are using clownfish because we want to manipulate the hypothalamus to see how that affects the sex changing process so we want to insert designer receptors so we can artifically activate or inactivate certain cells and see how that affects things like E2 or 11KT production among other things.

Thanks for the explanation. Since it seems like the injections are the problem you should try to confirm that with some controls. If it is the injections then you're going to have to try to get your DNA in some other way. Look into diffusion techniques or some other type of membrane disruption that isn't as invasive as a needle.

 

[c_healy]

I haven't, do you think high aeration during incubation could be harming them? My reasoning was it keeps the eggs clear of debris and microorganisms as well as keeps them oxygenated.

As you know they’re very delicate. It’s possible that’s causing some issues and worth a try on the next clutch. It doesn’t take much to kill them.