gaberosenfield
Guest
I wasn't accusing you of referring to it as an infection @IOnceWasLegend! I just wanted to make it clear to others that the link you provided did not give any evidence of an infection.
I'm not sure of the exact number of coral genomes that have been sequenced, but we have sequences for at least a dozen species and more are being published every month. The cost of sequencing has come down so much that many groups are sequencing corals all around the world. Pretty much all the fluorescent colors we see in corals under actinic lights are the results of fluorescent proteins, many of which are closely related to GFP.
For the non-biologists reading this, I apologize for the jargon in the rest of this post!
The Pringle lab at Stanford and others have been working to modify the genes of corals, and it is difficult for many reasons. Of course, you do have to design CRISPR guides individually for every site you want to target in each genome. But this isn't the difficult part. So far, no one has been able to successfully integrate foreign genetic material into a coral or anemone genome. We can knock out genes in corals and anemones, and we can transiently express foreign genes in anemone larvae, but we haven't been successful in permanently adding genes to cnidarian genomes. This kind of work is difficult for two reasons:
1) Methods for getting genetic material into cnidarians are limited. We don't have viral transfection vectors like the ones researchers use to add foreign genes to human or mouse cells, and many of the other transfection systems/reagents (e.g. lipofection systems) don't work in full-strength seawater at pH 8. Furthermore, we can't culture cnidarian cells ex vivo, so we have to transform naturally produced single-cell zygotes using either electroporation or microinjection. All attempts to transform adult Aiptasia in the lab have failed. We think this is because the mucus coating these anemones has very high DNAse and RNAse activity, meaning any unprotected genetic material is destroyed before it can get to the anemone cells. I've seen no reports of attempts to transform adult corals, but presumably their mucus also has high nuclease activity.
2) We haven't been able to make mutant lines of cnidarians. Given how difficult it is to get corals to sexually reproduce in captivity, attempts at genetic manipulation are restricted to the brief windows of annual natural spawning on the reef. We can get Exaiptasia pallida to spawn weekly in our lab, which is one of the main reasons we are working on Aiptasia rather than a coral. However, we can't get the Aiptasia larvae to settle and metamorphose into adult anemones, which means even if we successfully manipulate their genomes we can only do experiments on the manipulated larvae for a few weeks before they die. So we can't make stable mutant lines of E. pallida. We probably can make stable mutant lines of corals since many of them are easier to settle and metamorphose in the lab, but we have few opportunities to try this since we can't get them to reliably spawn in captivity.
But we are working on these problems. Before the SARS-COV-2 shutdown, I was just about to try Agrobacterium tumefasciens-mediated transformation on adult Aiptasia anemones. A. tumefasciens is a bacterium that naturally infects the stems of many plant species. However, it has the unusual ability to literally inject a special piece of DNA, known as the tumor-inducer (TI) plasmid, into plant cells. Once in the plant cell, the TI plasmid expresses a number of tumor-inducing genes and integrates into the plant cell's genome. Infected cells form a large tumor or "gall" on the stem of the plant, and the A. tumefasciens cells live inside this tumor. There's a lot more to that story, but it turns out this bacterium can inject the TI plasmid into pretty much any eukaryotic cell, including fungal, human, and even sea-urchin cells. So I modified a version of the TI plasmid to encode GFP in a form that Exaiptasia pallida can a express and I put this modified TI plasmid back into the bacteria. Since the TI plasmid is protected by the bacterial injection apparatus, this method should avoid the nuclease activity in adult anemone mucus so it should work in both larvae and adults. I was planning on starting my A.t.-transfection experiments the day we were shut down
.
We are also trying to us CRISPR/Cas9 to tag several genes in E. pallida with GFP at their endogenous loci. We were ~1 week away from these experiments when we were shut down...
I've also been trying to get E. pallida cells into culture since January of this year. Marine invertebrate cell culture is notoriously difficult, and so far I haven't had any real success.
Below are two gifs of a live 2 day old E. pallida larva, magnified 200x. They use tiny hairs called cilia all around their bodies to rotate and swim (you can see these if you look closely around the edges of the larva). The tail-like thing at the top is not a flagellum, but an "apical tuft"; we don't know why they make them and they swim with these tufts facing forward. The mouth is at the lower end and the darker interior of the larvae is its gut. The first gif shows the larva under white light (DIC), and the second gif shows the same larva under blue light that excites GFP fluorescence. E. pallida doesn't encode GFP in its genome; this is the first larva that I personally transfected (via electroporation of zygotes) with a plasmid encoding GFP. Unfortunately, the GFP is only expressed transiently, and only in a stripe along one side of the larva. This larva went dark within a week.
Other members of my lab have successfully gotten larvae like this one to transiently express cyan and red fluorescent proteins, as well as a fluorescent protein called kaede that starts off green but can be permanently switched to red if exposed to UV light. Of course, we've also expressed other proteins and siRNAs for research purposes. Side note: kaede is a naturally occurring fluorescent protein from the open brain coral (Trachyphyllia geoffroyi), so these corals may change colors quickly when exposed to UV light.
I'm not sure of the exact number of coral genomes that have been sequenced, but we have sequences for at least a dozen species and more are being published every month. The cost of sequencing has come down so much that many groups are sequencing corals all around the world. Pretty much all the fluorescent colors we see in corals under actinic lights are the results of fluorescent proteins, many of which are closely related to GFP.
For the non-biologists reading this, I apologize for the jargon in the rest of this post!
The Pringle lab at Stanford and others have been working to modify the genes of corals, and it is difficult for many reasons. Of course, you do have to design CRISPR guides individually for every site you want to target in each genome. But this isn't the difficult part. So far, no one has been able to successfully integrate foreign genetic material into a coral or anemone genome. We can knock out genes in corals and anemones, and we can transiently express foreign genes in anemone larvae, but we haven't been successful in permanently adding genes to cnidarian genomes. This kind of work is difficult for two reasons:
1) Methods for getting genetic material into cnidarians are limited. We don't have viral transfection vectors like the ones researchers use to add foreign genes to human or mouse cells, and many of the other transfection systems/reagents (e.g. lipofection systems) don't work in full-strength seawater at pH 8. Furthermore, we can't culture cnidarian cells ex vivo, so we have to transform naturally produced single-cell zygotes using either electroporation or microinjection. All attempts to transform adult Aiptasia in the lab have failed. We think this is because the mucus coating these anemones has very high DNAse and RNAse activity, meaning any unprotected genetic material is destroyed before it can get to the anemone cells. I've seen no reports of attempts to transform adult corals, but presumably their mucus also has high nuclease activity.
2) We haven't been able to make mutant lines of cnidarians. Given how difficult it is to get corals to sexually reproduce in captivity, attempts at genetic manipulation are restricted to the brief windows of annual natural spawning on the reef. We can get Exaiptasia pallida to spawn weekly in our lab, which is one of the main reasons we are working on Aiptasia rather than a coral. However, we can't get the Aiptasia larvae to settle and metamorphose into adult anemones, which means even if we successfully manipulate their genomes we can only do experiments on the manipulated larvae for a few weeks before they die. So we can't make stable mutant lines of E. pallida. We probably can make stable mutant lines of corals since many of them are easier to settle and metamorphose in the lab, but we have few opportunities to try this since we can't get them to reliably spawn in captivity.
But we are working on these problems. Before the SARS-COV-2 shutdown, I was just about to try Agrobacterium tumefasciens-mediated transformation on adult Aiptasia anemones. A. tumefasciens is a bacterium that naturally infects the stems of many plant species. However, it has the unusual ability to literally inject a special piece of DNA, known as the tumor-inducer (TI) plasmid, into plant cells. Once in the plant cell, the TI plasmid expresses a number of tumor-inducing genes and integrates into the plant cell's genome. Infected cells form a large tumor or "gall" on the stem of the plant, and the A. tumefasciens cells live inside this tumor. There's a lot more to that story, but it turns out this bacterium can inject the TI plasmid into pretty much any eukaryotic cell, including fungal, human, and even sea-urchin cells. So I modified a version of the TI plasmid to encode GFP in a form that Exaiptasia pallida can a express and I put this modified TI plasmid back into the bacteria. Since the TI plasmid is protected by the bacterial injection apparatus, this method should avoid the nuclease activity in adult anemone mucus so it should work in both larvae and adults. I was planning on starting my A.t.-transfection experiments the day we were shut down
.We are also trying to us CRISPR/Cas9 to tag several genes in E. pallida with GFP at their endogenous loci. We were ~1 week away from these experiments when we were shut down...
I've also been trying to get E. pallida cells into culture since January of this year. Marine invertebrate cell culture is notoriously difficult, and so far I haven't had any real success.
Below are two gifs of a live 2 day old E. pallida larva, magnified 200x. They use tiny hairs called cilia all around their bodies to rotate and swim (you can see these if you look closely around the edges of the larva). The tail-like thing at the top is not a flagellum, but an "apical tuft"; we don't know why they make them and they swim with these tufts facing forward. The mouth is at the lower end and the darker interior of the larvae is its gut. The first gif shows the larva under white light (DIC), and the second gif shows the same larva under blue light that excites GFP fluorescence. E. pallida doesn't encode GFP in its genome; this is the first larva that I personally transfected (via electroporation of zygotes) with a plasmid encoding GFP. Unfortunately, the GFP is only expressed transiently, and only in a stripe along one side of the larva. This larva went dark within a week.
Other members of my lab have successfully gotten larvae like this one to transiently express cyan and red fluorescent proteins, as well as a fluorescent protein called kaede that starts off green but can be permanently switched to red if exposed to UV light. Of course, we've also expressed other proteins and siRNAs for research purposes. Side note: kaede is a naturally occurring fluorescent protein from the open brain coral (Trachyphyllia geoffroyi), so these corals may change colors quickly when exposed to UV light.
