Fluorescent & luminescent modification

In relation to a question posed in the slack, true bioluminescence is caused by reactions within the cell. Most animals that appear to be luminescent actually use colonies of luminescent bacteria to create their glow. This is a basic summary of bacterial bioluminescence, and while you're welcome to read it in depth, the major takeaways are that the light emitting mechanism is an oxidation reaction, and that the majority of the bacteria that are not obligate symbionts are parasitic. The gene sequences that the bacteria use to emit light are commonly referred to as the lux genes. There hasn't been much publicized research into how well the genes express themselves in eukaryotic cells, except for this study on modifying baker's yeast. There have been studies done on inserting firefly luciferase into mammalian cells, nothing has been done with the intent of seeing the effect on a living organism.

Fluorescence on the other hand has been much more deeply investigated, due to the ease of use of the jellyfish GFP gene. This is the "glowing" gene that has been successfully put in cats, rabbits, and that you can buy at-home kits to play with from various vendors, including The ODIN. I'm sure everyone's at least passingly familiar with it, and how blue light causes it to appear to glow green.

As to uses and purpose, obviously we're mainly looking at cosmetic modification, so you'd want it in your skin somehow. There have been successful in vivo gene editing procedures. Those could be used to modify the skin, but then you run the risk of hitting the wrong area, or having leakage to cells you didn't want modified. Not a big deal, unless you're wanting some sort of design. For that, I would expect grafts of modified skin to be more effective, as you could grow the skin from however many modified cells and trim it into whatever design you wished.

Growing the skin separately to test any modifications first is of course ideal, all that's needed is a standard crispr kit and a bioreactor.

Comments

  • couple things.
    1, you can't do bioluminescence in mammalian cells if you're using any of the bacterial pathways. some of the compounds in the synthesis pathway for bacterial luceferin resemble molecules that normally are only present inside mitochondria. If you body sees them, it thinks your mitochondria are exploding and panics and the cell self destructs. A friend found this out the hard way when testing on fish. Ended with a bunch of goo-ified fish. Not good. Look into the p. stipticus pathway. I believe a construct for that pathway is circulating. may be a good place to start. And in so far as fungi are basically sesile animals you'll have a better chance of not exploding.

    1. As to growing skin, hard no. you are VASTLY underestimating what it takes to do that and no, crispr and a bioreactor ain't it. Try 50k worth of hardware and an entire mammalian lab. Mammalian cell culture is both expensive and easy to screw up and it's not something you should jump into without training or you'll just be burning money. Lots of it.

    2. You're far more likely to succeed with simple fluorescence. There's already prebuilt plasmids that do that on addgene. A few mods to the sequence can make it more permanent and getting it into your cells is easy. no crispr required. Also I don't think cripsr means what you seem to think it means. It's not magic, and it's not needed to modify cells. To get the DNA into cells, lypofectamine or PEI are the go-to. Mix it with some DMSO could help get it across your skins outer layers and into your cells but expect the gene to spread a bit locally due to diffusion of lyposomes before absorbtion into cells.

    3. but in so far as you don't already know all this, you should probably learn a lot more biology before messing with your DNA. it's not a joke and it's not to be fucked with if you don't know what you're doing. Take it very seriously and give it the respect it deserves or run the risk of very seriously fucking yourself up. People are getting all excited about gene mods and wanting to just jump in cause they saw others do it. IT'S NOT A GOOD IDEA unless you know what you're doing. Same thing I'd say to kids wanting to build Fusors. You're more likely to end up dead than produce fusion unless you actually know and mitigate the risks.

    1. You're right, I missed that on the bacterial pathways, I couldn't find any research on their introduction into mammalian cells at all. However, the firefly luciferase genes from P. pyralis have been successfully inserted & expressed in mammalian cells.

    2. Human skin has been grown on a fibrin scaffold in constant-flow bioreactors, with a fair degree of success. Obviously in laboratory settings, but the point remains that it can be done if someone wished to attempt it.

    3. I agree with you, and while I brought it up, fluorescence is not what was asked about, which is why I didn't touch on it much.

    4. The examples I listed were simply proof that in vivo modification works. Lipofection, like all other commercially available transfection methods, is intended for use in a setting where the cells are inundated in a solution, and none of them suggest good results when attempted on a living organism. Also, Mirus' TransIT stuff has shown better efficiency than Lipofectamine.

  • I apologize for my earlier tone. I hadn't really considered any commercially available transfection methods for the reason I stated above. It's not that I don't know all that, I have a tendency to get tunnel vision when I'm looking at an approach to a problem.

    Thinking about it a bit more though, if the liposome solution could be thickened a bit to prevent diffusion, it could be a way to 'tattoo' glowing ink on. Something to test on a pig in the near future, I think. At least with the GFP gene.

  • edited September 20
    I was looking at that a while ago, and I think that the dual system luciferase seems to be the best route.https://www.promega.com/resources/protocols/technical-manuals/0/checkmate-mammalian-twohybrid-system-protocol/
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