Gene Electrotransfer

Some light reading on the subject: Electroporation  and Gene Electrotransfer .  Here is also a video on a related procedure.

Ladies and gentlemen, I believe we've found our first cheap and reliable gene therapy "vector".  All we need to do is get the electroporator built.  
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  • Lets do this. Any of our electric people know how complicated this might be?
  • if you can give me the parameters like voltage and pulse duration etc. piece of cake.
  • IanIan
    edited December 2011
    I agree with @ThomasEgi; this is pretty simple as far as electronics go.

    The specific voltage depends on the type of cell membrane used, and the pulse duration and shape (square vs. sinusoid, etc.), but typically, from my understanding, the rule of thumb is about 10,000 V/cm for the applied voltage per unit length of the cell, with square pulses of about a ms or so.  This course website seems to have a fairly good procedure on it.

    ~Ian
  • 10kV per cm? that is quite an electric field you get there. you better make the electrode-spacing very small so you can get the voltage down. 1mm would be quite reasonable. mechanically not much of a problem.
  • IanIan
    edited December 2011
    Yeah; I was looking at electroporators that are sold commercially, and they usually have something like a few hundred volts across a gap of a couple mm, with pulses of a few ms.  I'd suggest getting a commercial one and taking a look at it, but those cost thousands of dollars.  Also, the 10 kV/cm would be for the smaller cells, such as bacteria; I think something like a yeast cell would be more like 100 kV :/

    EDIT:  fixed my screwed-up calculations
  • I realize that this is an old discussion, but I'm trying this anyway.  The first iteration is going to be simplified and generalized quite a bit as a proof of concept.  Later generations can be more tuneable for specific cell types.  Here's what I'm thinking...

    AtTiny85 or AtMega328P to control the pulses (I use an Arduino as ISP to program the chips to save money, but you could easily use an Arduino or compatible... if the AtTiny85 use an Adafruit Trinket, or an Arduino or Teensy (I love this little board, by the way - hell, my ISP is built off of one.  Overkill, but cheap and small) if the code won't time correctly or is too much for the tiny to handle.

    The output from the MUX will either be implemented as a square wave using the Arduino tone() function, or analogWrite(), or digitalWrite() with the delay built into software.  1kHz - 5kHz seems to be the range, so I'll make this tuneable in that range - should be a trivial task.  I'm thinking just have a single momentary switch to step it up in steps of .25 or .5kHz and a small LED bar as an indicator.  The number of pulses will be set up the same way.  It looks like I'll be using the AtMega or a Teensy just for the pins.

    The output will drive a simple high speed transistor set up as an amp to then drive some high speed MOSFETs to do the power transfer to the electrodes.  For the moment, I'm assuming a 2mm spacing but that could easily change.

    I haven't completely figured out the power supply yet.  I have a rectified 5kV power supply and could potentially just use that with an appropriate voltage divider, or I could set up a transformer/rectifier closer to the output required (3.6kV as a lot of the documentation I'm finding shows this to be right in the range provided by many of the "professional" units).  

    Either way, my issue here is that I'm having a hard time finding data on the specific current used.  Once I have this information, I can design a current limiter.  My big concern is that as I step up the voltage, the current drops.  I'm concerned that I might not have enough current.  I strongly suspect that this is not the case, but still...

    My other concern is arcing.  At these voltages and the small gap, I could see this becoming an issue and reducing the viability of cells in culture.  I might have to rethink the spacing and increase the voltage, but that makes the current issue more likely.  As before, once I have data on the current required I can work out how much I can step up the voltage before this becomes an issue and figure out a more optimal spacing.

    Anyway, has anybody run across information on current in this kit?  I'm hoping so, as none of the information I'm finding has yet to shed any light on this.  Once I have this last variable, I don't think it will take long at all to get a prototype up and running since I have most of the components sitting on the shelf already.

    Also, did I miss where somebody has already successfully (or unsuccessfully, as that info would be helpful) built one?  No sense in reinventing the wheel if there's a cheap solution already out there.
  • edited September 2014
    Alright!  Better data on electrical characteristics.

    For mammalian cells, 4mm gap @ 6.25kV/cm... That's 2.5kV - easily achievable.

    As far as current goes - most earlier models used 400mA, but recent studies show greater cell viability and highly effective transfection at 125 µA.  Again, this is easily done.  I am also finding information on successful transfection at 1-5 µA, although at much lower levels.

    Hopefully I'll have a preliminary design within a week or so.  It's going to take a bit longer than it otherwise might, as I'm trying to do this with as many components that I happen to have on the shelf.

    If anybody has potentially useful information, I'd love to have it.  If not, I'll post here when some progress is made.
  • What's the goal here? I ask because I work in a research lab where we routinely electroporate chick embryos, and it being "electrically simple" doesn't mean that it isn't otherwise tricky. I might be able to provide some insight, or at least try and dissuade you from pursuing non-fruitful paths.
  • seconded.  what are we looking at doing here?designing and building a tool cheaper than you can get it at a universi surplus sale, or...?

    also,the op here talks about gene therapy vectors. you guys know you cant like... stick your finger in one of these,right? what's the project? and if it is some cool gene work, why rebuild the wheel to do it?

    if youre just planning on doing bacterial work, cold shock is still a totally solid and consistantly successful technique.
  • edited October 2014
    It's a proof of concept for a cheaper than surplus device.

    Ars artis gratia.

    That being said, if @dokclaw has any suggestions as to what cell types to start with and what is feasible in a longer view, I am all ears.
  • Commercially available electroporators are generally either cuvette-based, or have hand/clamp held electrodes. The area that is actually electroporated is really, really small. Maybe 5mm2 MAX with the hand-helds (and I couldn't say what the electrical requirements of larger electrodes would be... Probably pretty burny.) This is why I ask what the goal is. I don't think that there's necessarily value to building a proof of concept device unless you have a clear end goal for that device. I will elaborate.

    These things work by putting DNA next to a cathode, and the cells to be electroporated next to the anode; unless you can get the cells of interest between the electrodes, you're not going to have much success. If you're thinking about (say) tattooing with one of these things, there's going to be a lot of skin-clamping that has to go on first to get the electrodes to straddle the tissue. That's to say nothing of the voltages required to actually get nucleotides into your skin. And also the fact that the cells you would be electroporating would be only the most superficial skin cells; the dead ones that are unable to express your gene of interest. 

    I would suggest that, at least initially, you try and build something that is able to electroporate cells in a cuvette. In these cases, the walls of the cuvette are the electrodes, and you mix cells and DNA in a liquid medium that sits in between the electrodes and zap them. Some proportion of the cells will take up the DNA (the latest ones advertise as high as 99%, but these are optimised conditions, cells, and plasmids, run by individuals whose only job is to get that number as high as possible; numbers are frequently as low as 10-30%). 

    As to the cell type, you might as well start with yeast. It's easily obtainable, and relatively easy to handle (not sterile technique so much as aseptic technique... Bunsen burner going, etc). You still need things like a centrifuge, though you can rig one up with a bike wheel, I'm sure. You would also need to get a plasmid so you can express something like beta-galactosidase to check how efficient your electroporation has actually been, and preferably some kind of selection factor (antibiotic resistance is common in bacteria, but I don't know how people select for transformants in yeast...)

    Here's an example protocol for transforming yeast:

    It's really, really easy to kill cells with electroporation. We've been using the same protocol for years. I think the same one was being used 20 years ago, with the current properties being modified ever so slightly. We use hand-held electrodes (platinum/iridium) 5 x 50ms pulses of 30V, separated by 1s, the electrodes held ~4mm apart. The electrode separation is critical here; a lot of cells will be killed if they are too close to the electrodes, and if they're too far apart, the efficiency is terrible.

    In the long term, I don't think it's really going to be feasible to express genes of interest in human tissues directly; the epidermis is quite a good resistor from what I understand, so the voltage requirements for the current would be pretty high. This is in addition to the issues brought about by having to have the tissue clamped between 2 electrodes, and only electroporating the superficial layers. I guess you could have a clamp (like a piercer's clamp), two wet , malleable, conductive pads that contain the electrodes (like ultrasound) and then inject the DNA construct into the subject, between the clamps before zapping them. You'd need a pretty brave volunteer for that!

    That said, expression of genes in cells derived from a subject is not unfeasible. Companies like celltran (http://www.celltran.co.uk/#) are able to grow skin grafts from burn patients, and re-introduce them successfully. If you were to insert an electroporation step into the process via the cuvette method (as suggested with yeast), then you could express what you wanted to in the skin, though the subject would also have to have a wound in that are for the graft to take. People do scarification and cosmetic burning all the time though, so it's not like this is unheard of...
  • Hey,

    So I am curious as to the genes you are looking to insert/delete. I am an undergrad working in a fungal genetics lab and electroporation is something I am familiar with. In molecular lab we used it for things like adding antiobiotic resistance to E. coli or GFP to yeast.

    That said, I would not recommend using Electroporation and Cell membrane disruption as a method to modify a mammals DNA. I believe it was done in OP's article purely out of desperation.

    @dokclaw , You are actually the reason I made an account. (Apart from sticking a magnet in my finger Saturday) It is good to see another research biologist on here. I like your point about the skin grafts, and can see electroporation having potential there. Though I dont really know what you could do, besides adding some fluorescent proteins or pigments, which could be cool. 

    However, we are in the midst of a revolution. A CRISPR CAS revolution. 


    The above paper describes a procedure for deleting/disrupting genes in a living adult mammalian brain. 

    A brave and clever soul could create a transformation vector to be injected into the human body. I can think of a few things to add, like a calstabin gene, or GFP. A fluorescent protein encoding gene is probably the way to go as they can be non-lethal(see glo-fish) and their expression only noticeable under uv light. 

    This could be done for 1000-6000 dollars if the right equipment was already available. The question is, who will be the first to do it?

    One more point I would like to make about electroporation, with the human micro biome project ramping up our knowledge of the bugs inside of us is increasing rapidly. These microbes are definitely manipulable with standard electroporation procedure and genetic insertions could do anything from allowing you to digest cellulose/eat grass like a cow (but prepare for nasty bathroom effects) to producing HGH in vivo. I think this topic deserves its own thread though.
  • hmm. the crispr cas method is the closest thing i've seen to a method i've been developing to deliver new genes to a living host. Gonna have to read up on it. Also if you can only think of 2 genes to add to the micro biome you're seriously lacking in creativity :P Off the top of my head I can think of a massive array that i'd love to add in. For example, there's a gene that allows for resistance to arsenic. Think about what'd happen if your whole gastro tract was covered in bacteria able to deal with arsenic. Or what about adding in genes to produce all the vitamins we currently have to EAT. If it was just produces instead? that'd be amazing.
  • edited November 2014
    I shall enter with my customary wet blanket. *Drip, drip*.

    Genes that encode resistance to arsenic in bacteria will allow the bacteria to survive arsenic poisoning, not the host. 

    Additionally, the introduction of foreign bacteria or genes into the gastrointestinal system would have so many unforseen consequences. Let's say you are able to insert a series of genes that allow vitamin D synthesis into lactobacillus (it's not going to be just one gene) .Those bacteria are now less energy-efficient than the other lactobacillus in your gut, because some of the metabolic energy they were using to keep themselves alive is now being used to produce vitamin D, so they be out-competed and die. Unless you can give bacteria a competitive advantage, they will die out in your gut pretty quickly.

    I guess you could try and squash some additional genes in there that allowed those bacteria to efficiently metabolise and get energy from something that you hadn't commonly eaten until that point, then introduce that into your diet. That would provide at least a short-term boost to survival to your pet bacteria, at least until some others learned how to metabolise the foodstuff as well. 

    The microbiome is an ecosystem, and we don't have a fucking clue how to manipulate a whole one with good success. I guess at least with the gut you can flush it out and repopulate it if it gets too nasty...
  • Never said it would give you resistance to arsenic, just your gut. So when you die of arsenic poisoning you have a nice healthy gut :D Jeez.

    Also the addition of one gene does not a disadvantage make. If you only have the produce it in low levels it doesn't come as a disadvantage. Also after enough exposure horizontal gene transfer would share the gene around so you'd always be producing it. Yes it may be an ecosystem but we screw with it all the time. Every time we take antibiotics the whole thing goes to shit anyway. Why not throw a new creature into the mix and see what happens? Worst comes to worst it dies out. If it's weaker it won't overtake the whole system anyway. And ya it's got unforeseen consequences. So does sticking a chunk of heavy metal into your hand or taking a whole clusterfuck of noots. That's why we test it and see what happens. That is how science works. If we didn't do things because of the possibility of unforeseen consequences we'd be living in the dark ages. Not to say we should go barreling forward, but it could be interesting to see the affect on the body.
  • I know that the ethos of this forum is one of DIY science and trying things out to see if they work, but when it comes to genetic modification of ourselves or our microbiomes I am incredibly cautious about it, and I don't think that anyone will ever be able to convince me that my caution is misplaced. I'm fully behind things like Circadia because it is essentially an inert , implanted lump; it can't replicate, perform horizontal gene transfer, etc. etc. 

    Any time you fuck with the human body there are consequences. "Sticking a chunk of heavy metal into your hand" would have had a whole ton of consequences if we didn't know about how to coat the magnets beforehand so they didn't cause an immune reaction. And "Worst comes to worst" is not something that you can say when applying it to a complex (as in the mathematical definition) ecosystem, because by its very nature you cannot foresee what will happen. When people implant, they are pretty confident what will happen, because doctors have been putting foreign objects into people for over 100 years, and have got better and better at it.

    That said, if you're interested in vitamin synthesis and how to induce it in bacteria, take a look at this site: http://www.kegg.jp/kegg/pathway.html 
    It contains pretty much the sum of human knowledge of genetic pathways in a huge number of organisms. Lactobacillus acidophilus already contain the synthetic machinery to make B12 (and likely others), and are already used for its industrial production, so you can at least know that the organism you want is capable of producing it. Of course, to increase its production, you'd have to tinker a lot with the genes of the bacteria (likely already done to save money in industry) and diet of the host, then find a way to get the vitamins out of the bacteria, possibly by packaging them into vesicles for export (I don't know how you would do this with small molecules, only with proteins, you'd probably have to look at pathways in secretory cells). 
  • Well thanks for the link. And even if you don't want to test it (don't think im crazy enough to try myself either) you don't have too. I just wanna design and make some interesting bacteria and eventually test it on an organism and see if it has benefits. If something simple like a vitamin could be produced and maintained then who knows, it could lead to other advancements. 
  • Oooh! I'd test it out! Not sure how you'd get them into my gut, but I would be so down for that. B12 is like, so important, and I'd love to experimentally be self sufficient in it because my diet is incredibly terrible.
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