Transfection methods – Which is best?

Several basic types of transfection: gene gun (effective on almost anything), electroporation (oldie but goodie), liposomes, polymer complexes, and viral particles, with or without reverse transcriptase.

Cell culture transfection

Liposomes are sometimes problematic, because they kill cells, but these days there are kits from Sigma-Aldrich and others that are quite good. Polymer complexes like Turbofect from Thermofisher also work well, distinguished from liposomes in that they cling to the DNA, and work just about as well with large DNA strands as with short plasmids of a few thousand base pairs. But nobody really cares about that, you just want your DNA to get into the cells so you can move on. Usually, in cell culture, those are just fine.

There are electroporation systems that work in cell culture for both adherent and non-adherent cells. It typically takes a little more handling, and if you don’t set equipment right, you’ll smoke your little cells. But I’ve found electroporation to work fine, even when things are a bit off. Cells are pretty tough if you keep them cold, etc.

The nice thing about gene guns is that you can use them easily right on a plate. Bang, it’s done. They don’t do so well with suspended cells, because the particles won’t penetrate much into water. But usually you can get around that by spinning and draining cells, or culturing them on plates the way it’s usually done when creating monoclonal antibodies. Sterile technique is a lot trickier then though. You’ll need to take great care with your facility, use UV lamps and wipedowns all the time.

Not so many people use viral particles for cell culture, and that’s something I don’t have experience with, so I won’t say any more about it.

In vivo transfection

All of the above work in-vivo also, although you have to prep a bit differently. For instance, to use a gene gun with a plasmid into muscle tissue, you’ll have to open the skin and lay bare a enough muscle. You’ll need to keep the distance correctly, and may want to protect non-inoculation areas from getting transfected. Aluminum foil will do fine surrounding the area if you care.

I like electroporation into muscle also. This is done with a needle surrounded by fine tines that surround the needle in the muscle. The needle becomes negative terminal and the tines around it positive, to drive DNA into cells. This method has been shown to work well, at least in large animals.

In addition to these methods, I like using microbeads of 2-5 microns composed of PEG, PLG or something like that, with your DNA embedded into it. That’s an optimum size for uptake into cells. The drawback is that this can generate an immune inflammatory response – mild, but that needs to be compensated for. (And Butterfly Sciences has some vehicle formulations for that.)

I think that injection into mouse muscle is difficult. Their muscles are so small, and syringes are large enough, that I think it’s pretty easy to screw up the injection. So I’d prefer not to work with anything smaller than a rat.

Special notes about viral particles

Viral gene therapy is more widely known than bare DNA plasmid therapy. In this method, a recombinant virus is engineered containing a gene of interest. Adenoviruses, adeno-associated virus and lentiviruses are commonly used. Viral vectors can be made specific to a tissue type by careful engineering of the virus’s attachment moities.

This method has several drawbacks:

  • The doses required can cause death through a sepsis-like syndrome, although animals are less likely to suffer this consequence because their immune systems are more robust than humans. Jesse Gelsinger died this way. If you get into this kind of trouble, try a shot of Enbrel.
  • There is some risk of cancer. It may be low, but it is there. AAV has a low rate of integration into chromosomes, very low. But it’s not zero.
  • With retrovirus deliver of genes the risk of cancer is significant. Around half of all the children given gene therapy for SCID died as a result.
  • Viral delivery of genes is irreversible. Viruses disseminate in the body, and cannot be removed surgically. In theory, in some limited cases, like synovial fluid, the viral vector should not get outside the synovium. But what about injection errors? Those happen. With plasmids, there is a small section of muscle where the transfection is virtually entirely localized.
  • If the viral vector is accidentally injected into the wrong person, you have a serious problem. Of course, that shouldn’t happen, but when thinking about products that will be sent out all over, things happen. If it’s an animal product, someone might use it on a human. Medical errors happen all the time, from amputation of the wrong leg, to administering the wrong dose of medication. Count on it happening sooner or later.

Both viruses and bare DNA (plasmids) are workable. I prefer the plasmid system because it’s more forgiving. Transfection efficiency is not as good as viral delivery, but it’s good enough.