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Gene tattoos

BMedSci

April 2015

edited April 2015

in Genetic and biology based mods

What I'd love to do is use genetic engineering to reversibly modify yourself. The context I thought it could be used in was temporary tattooing that would last a few years. You could do this by inserting a colour gene (I was thinking GFP (gree fluorescent protein) as a classic example) into the non-dividing cells in the skin and allowing it to be expressed, thus giving the skin colour. You could get some awesome glow in the dark features (I was thinking maybe like an ET-eske finger flashlight). Since it would be in the non-dividing layer it would eventually go as the expressing cells died off, but if it was put into the deeper dividing cells you could insert an inactivating gene alongside to turn it off should you ever want to (or insert a sequence into the gene to fragment it). The vector could be a simple adeno-associatedvirus or better still simply encase the gene in a liposome and rub it into the skin (as is done for some other gene therapies). You'd have to removed a few skin cell layers such as the stratum corneum but my professor said it should be feasible but I'd get shot down by ethics councils.

The main issues lie with the gene inserting into somewhere where it could disrupt another gene's expression and potentially cause cancer. To avoid this I'd make it have a very low level of expression in case it did end up in a promoter region, so that it would minimise any excess gene promotion.

What do you think? Feasible or crazy?

Also, does anyone have the tecchie know-how/means/balls to do this?

Tagged: tattoo, gene, vector, DNA, RNA

Comments

Displaying all 15 comments

glims

April 2015

Please dig around a bit in the tetrachrome, adeno-associated virus, and other threads. This type of thing has been mentioned a few places.

I like the idea of being able to pull the ideas out of all of them and actually do something tho
BMedSci

April 2015

Yeah, I've been told that a few of these ideas had been floated around.

I don't know if this has been suggested but I think that inserting RNA inside a liposome packaging would be a good proof of concept if you have any idea how to do such a thing?

On another note, this would more than certainly cause an immune reaction (albeit probably a small one) so that's another safety issue.
chironex

April 2015

I guess since genetics is my thing I should jump in here. in reverse order, feasible, maybe at best. Crazy? absolutely but then again have you seen what else we do around here?. Your best bet is some sort of plasmid. Your best delivery would either be as you say a liposome or to avoid the random spread of new dna, a nanoparticle delivery system. I've been looking into a few but due to school and life getting in the way my research into that has been... delayed slightly. Viruses honestly suck for this as you need to take a fair amount of immune-suppressants for them to work well and I don't think i need to explain why that's a shitty idea. As entertaining as this whole idea is, something I'm not sure most realize and I confess I didn't really until I got onto this board was the scope of work needed to make something like this work. Does that make it impossible? no. Just means a lot of prep work before we hit the lab. So to get started you need more than a vague idea. You need an exact protein you want produced. You need the exact cell type you want to target and where you want it expressed and in what volume. You need to design the control system. You need to make sure the DNA will migrate to the nucleus to make sure it lasts for a good long time. You need your delivery vector. so, pick all of that and we can go from there. Start with what protein you want and where you want it. Also is it going to be administered like a typical tattoo? If so you'll need a lot of your plasmid which will have to be amplified and encapsulated. all of which is doable but not without the preplanning.
BMedSci

April 2015

Nice, I'll do some serious research into this in my spare time (and probably mostly after exam season).

So far I'm thinking this

-Protein=GFP (though might look into others/modification of GFP to stop it being degraded)
-Delivery=liposome

This is where I don't know what would be best:
-Nucleic Acid=pure RNA because I don't want to risk insertion. The length of time the glow is expressed can be dependent on the protein and not the RNA, which I would rather see degraded ASAP after making being translated as having bits of DNA/RNA floating about doesn't seem too safe.

-Plasmid=I have no idea. I know GFP can come in a pGLO plasmid, which I guess already has the expression cassette in-built and doesn't kill bacteria so I assume its safe-ish. I guess pGLO would be the easiest solution as I have no experience with expression cassettes.

-Administration= If its liposome delivered I assume all you need to do is remove the top layers until you get to the cells you want to target (I'd say any at this point because I don't know) and then rub it in as a cream.

Issues: I have no clue how to ensure migration to the nucleus, but if RNA is used then I don't think this would be a problem. The RNA would probably also avoid any Toll-like-receptor (TLRs) that would be liable to cause reactions to bacterial DNA (which I assume would cause red flags for the plasmid);

Do you guys know where a total newbie would start? I'm not a stranger to the lab due to my degree (I've done a fair bit of DNA stuff but only in a bacterial context but I'm sure it doesn't really transfer).

Thanks guys :)
chironex

April 2015

liposomes are old tech that don't work that well. Try something along the lines of a copolymer diblock nanoparticle or zwitterion diblock.

As to your gene of choice, gfp is sorta boring and you'll need a uv light for it. If you're modifying your dna you want something better than that. I'd suggest a luceferine lucefirase system or something along those lines. Means a bigger plasmid but that's not such a big issue.

The pglo plasmid is bacterial and won't work well in humans. you need to make the plasmid either from scratch or insert the genes you want into a ready made plasmid but those can run for a lot of cash. Rna breaks down hella fast so no point in using it. Random DNA doesn't usually get intergrated and if it's on a circular plasmid theres even less issues. Theres tags you can put on the end of the sequence if you want it to integrate but that's not needed.

So if you want to really do this first thing, dig into the protiens. go through the various types of glowing ones or colorful ones and then go from there. Personally i'd suggest a color changing thing rather than glowing. Glowing is ok but color change is easier and looks way cooler for times when you're not in the dark. although in theory you could do both. You'd need to design a control system that turns the first color off at a certain time of day or when you have certain levels of something in your body and triggers the next to start.
glims

April 2015

awww yeah, you can tell we've been talking, dropping the z-word ;)

basically, the point about the liposomes is that the term is very vague and you will probably break it down way before it gets to your delivery site. With your diblock you can use a liposome like model, but then you can also target it through chemical engineering of your hydrophilic end. Using somethign like a zwitter is good because they are usually biofriendly and by choosing the right carbon chain length, you can adjust the robustness of the nanoparticles formed.

This all avoids the main issue which chironex touched on which is once you get your nanoparticle into the correct area, how will you cause the gene transfer?. You mentioned bacteria and you are correct in that the gene work there doesn't really transfer. Bacteria have a tendency to take DNA and run with it. Our cells have a pile of mechanisms for destroying non native genetic material

AAV or Crispr is really your better bet here.
BMedSci

April 2015

Hey again,

I've come across something in my studies that could be a possible solution. It kinda seems very simplistic but apparently the application of naked DNA actually works to deliver DNA to topical areas (but that using something called microemulsion delivery system, which I think is the zwitterion method you're speaking of works better). I'm basing this on something I found in relation to melanoma immunotherapy (tumour antigen expression using plasmids) which cited this article http://www.eurekaselect.com/55323/article . You may wanna hit the article up for general knowledge but I think you probably know most of the stuff on it and it is fairly old (2006).

@chironex/glims
Any idea how to zwitterion it up?
With reference to the targetting I was hoping maybe a cream with the stuff in applied to scraped skin (to get rid of keratin layer) would suffice in getting it to the right spot.

@chironex
Your ideas about making colour changes to do with time of day are hella cool but I think that it would be waaaay above my pay grade to even think of something like that, though I think that if you could link it to insulin and/or glucagon levels you could make a cool sensor for blood glucose or diabetics.

Assuming I wanted to just go ahead and try this with a plasmid containing luciferin or some other bioluminescent molecule where would I actually buy one of these? If you guys don't know I'll ask around the teachers in my dept. but I'd rather not look like a mad scientist if at all possible.

Thanks :)
chironex

April 2015

Luciferin isn't a protein which means it needs a whole biosynthetic pathway to produce which means you need control mechanisms for every single step or you'll end up with a whole shitload of some precursor that for all we know could be toxic. This is what I mean by preplanning. I would start with a simple color. Forget the time of day thing. Literally pick a colorful protein, something who's sequence is nice and short. Or chose a compound with a short biosyntehtic pathway or a pathway already present you can pigyback on. Heme B has a structure similar to dinoflagelate luceferin for example. If you added the enzyme to use broken down heme to form your target colorful compound you'll be set. You'll have to tinker with the promoters and stuff on each gene to make it work if the enzyme needed come from plants or bacteria. As glims said the human body doesn't like foreign DNA so you need the DNA to look as native as possible. Not sure the best way of going about this but I'll look into it. You can't just "go ahead" with inserting foreign DNA into your body. If you screw around like that you can get really hurt. I'm not against this at all, in fact im all for it, just do this the smart way. We may look like mad scientists but we're at least careful. Also the chances of any ready made plasmids with GFP in them working with humans are slim.
It'll probably just be excised. That said a GFP derivative could also be a good start if you can find one that gives off a color when not fluorescing
Lex

April 2015

So bioluminescence? I have always thought glowing tattoos or bioluminescent markings would be really awesome to have. Though i have no idea how to do that genetically since that is not my specialty. But the final product is certain to look incredible.
BMedSci

April 2015

I think that the best way to go is probably GFP as a prototype since its been engineered into mice without killing them it seems like a fairly safe starting point in terms of not wanting to die and if it worked in mice there might be a chance that it would work for all mammals. Worst case I could email the lab that did it and ask if they think it might be human compatible.

Do you know how I could get the plasmids with the gene on (I could potentially make them by ligating the gene into it but that requires equipment, of which I have absolutely zero as of yet (and as I am not making any money since I'm a student that is likely to continue for a while))? Like where is the best place to source materials?

The reason I thought of using a cream to target it is that that seems to be a way that gene therapy is conducted in cancer chemo so I thought it might be a way since it works for that then maybe using the same protocols we could get the same results.
chironex

April 2015

I think I've mentioned this already. The chances of you finding a plasmid with everything you need built into it are slim. You honestly should be making the thing from scratch. You might be able to find a plasmid that will work and then you can insert the gene into it but this project is by no means one that can be hacked together in your bathroom. This is DNA, you can't just buy some rando plasmid with gfp in it, stick in your cells and hope for the best. Cream is fine, but again you need to pick and design your diblock and anything else that's going into that cream. This is a project i've actually been thinking about for a while so I may try it myself once my lab has a bit more funding for the necessary equiptment
U9Z0

April 2015

Just to derail this nice technical talk with a hypothetical - Would it be possible to make some sort of early warning tattoo? IE something that changes color if it encounters certain pathogens or cancer cells etc.
chironex

April 2015

Yes but that's something we can discuss once we have a framework built. Once we have the system to do this we can switch out the genes that would do something like that but ti's far more complex. We're starting simple.
BMedSci

April 2015

Ok, so let's say I want to make this from scratch I think this is my shopping list.

1: Plasmid that is big enough
2: Restriction enzymes that are specific for sites on the plasmid
3: Gene which has specific sticky ends for the restriction enzymes
4: DNA ligase
5: E.Coli in which I can multiply the plasmid
6; Sterile plates
7: Plasmid extraction kit
8: Various pipettes (which I know can run into hundreds of dollars alone) and tips
9: Agarose gel, kit, markers and power source
10: Something to turn the plasmids into zwitterions.
11: Cream

Is this a full shopping list and do you know where I can source at least some of these? What places do you use for buying kit/enzymes/proteins?

(I'll ask my uni next year for access to the labs maybe so that I don't need to buy all my own stuff)
chironex

April 2015

Ok lets try this again. Plasmid is just a ring of DNA. The point of NOT buying one is if you do you're getting all kinds of other random genes and code that will do fuck knows in the body. The point of making it yourself from scratch is that you know exactly what's in it and what'll interact with what and you've run all kinds of tests on it so you know how it'll react with the body.

You will need the gfp gene but that's easy enough to get since it's so commonly used.

You will need some restriction enzymes but theyre expensive as balls so be aware.

Dna ligase is easy to get comparitively

E. coli, you'll need a lab strain so you don't go infecting yourself with something nasty if you make a mistake. And some are designed specifically for this purpose. Not sure where to buy them off the top of my head but i can look around

plates are easy

plasmid extraction kit doesn't exist. it's a lab protocol. you'll need to learn it and all the equiptment needed for it. loads of different ways to do it

really the pipettes are where you see costs going up? that's the cheapest thing on your list but yes they're run you 50 bucks a piece usually not including tips

electrophoresis will be helpful so yes you'll need it

That's a whole bunch of chemistry so you need to pick which diblock construction you want to use and go from there for sourcing

your cream needs to be mixed specifically. You need something to keep your diblock nice and stable and help it penetrate.

you're missing loads of other stuff but start at the top and we'll keep working through it.

I wouldn't bother with the uni thing. Most professors get antsy about you using their gear, espceically if you mention you intend to stick new dna into yourself. I mean still try and ask but what'll likely happen is if you put in the work and come up with a fully realised protocol one of the labs on here may step up to do it for you. I for one would be happy to if you can do the work to make this a well thought out project since it's something i've wanted to do for years anyway.

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