We can do cool things if we can get nucleic acids (RNA or DNA) into cells. Everything from getting those cells to make specific proteins (by putting in the mRNA instructions or their DNA gene inserted into a vector) or stop making specific proteins (by putting in siRNA, etc.) But to do all that cool stuff you have to be able to get that DNA or RNA into the cells, a process called TRANSFECTION. There are multiple ways to do it depending on what you want to put in (how big, etc.), where you want to put it (type of cell (bacterial, insect, mammal?), location (in a dish, in a person?), and what tools your lab has access to! A few of the main methods include: heat shock with chemically competent cells, electroporation, and the use of cationic and lipid carriers. So let’s look at how they (hopefully) work and how we do them in the lab.
As I hinted at, you can transfect all sorts of nucleic acids, but what I typically want to put in are plasmids containing the DNA instructions for making my protein of interest. If you want to study how a protein works at the nitty-gritty-detail level like we do in biochemistry and structural biology, we need a LOT of it. And we need to be able to manipulate it. The instructions for proteins are written in the DNA language of genes and we can tweak a gene to tweak the protein and see what happens. We call this site-directed-mutagenesis and its really useful. Even if we don’t want to change the protein, just want to study its normal “wild-type” version, we’ll still need lots of it.
If we can get the gene into expression cells (commonly bacteria or insect cells), we can get those cells to make lots of it for us. Then we “just” need to purify it and run experiments with it. But we have to get the gene in there first…
The first step is to take the gene from its “natural home” and put it into a DNA “vehicle” called a VECTOR that the cells are comfortable copying and reading from. For bacterial expression, we usually put the gene into a circular piece of DNA called a PLASMID. There are different ways to do this, like “cut and paste” using restriction enzymes or “copy and staple” using SLIC. More here: http://bit.ly/2GQ06HL
That’s the MOLECULAR CLONING part. All that’s dealing with molecules in test tubes, but now it’s time to get real -> we need to get that plasmid we’ve engineered into actual cells. When we put DNA into *bacteria* we call this DNA-putting-inning TRANSFORMATION, BUT when we do the same thing, but put DNA into eukaryotic cells (cells with membrane-bound “rooms” like nuclei inside) – things like animal cells) we call it TRANSFECTION so it doesn’t get confused with tumor transformation (just gets confused with why its called something different…) Anyways…
To get it into cells you have to get past the cells’ membrane (and wall in the case of bacteria). Cells are surrounded by membranes made up of phospholipids which are “amphiphilic” molecules, meaning they have both hydrophilic (water-loving) and hydrophobic (water-excluded) parts -> they organize themselves into “sandwiches” called bilayers in which their hydrophobic tails huddle together away from the water and their hydrophilic heads are in contact with that water they so love – the inner heads are exposed to the watery cellular interior called the CYTOPLASM and the outer heads are exposed to the extracellular fluids.
Part of the reason the heads are hydrophilic is because they have negatively-charged phosphate groups (phosphorus surrounded by oxygens). Water likes charged things, (which is why salts like NaCl dissociate into Na⁺ & Cl⁺⁻ when you put them in water -> the water molecules want to coat each of these IONS (charged particles)). DNA is also negatively-charged, also because of phosphate groups (DNA’s backbone is full of them!)
Charged things might like water, but they don’t like same-charged things… positively-charged things (CATIONS) repel other cations and negatively-charged things (ANIONS) repel other anions.
Both DNA & the phospholipid heads are negatively-charged (ANIONIC) so, normally, the DNA won’t want to get near the membrane – which is a problem if you need it to get through that membrane… But, if you can hide DNA’s charge, maybe even mask it with positive charge, which the negatively charged membrane surface loves, you can get the DNA to bind.
Which brings us to the method I typically use for bacterial transformation: chemically-competent cells + heat shock. There’s LOTS more on this post: http://bit.ly/2Jj7L47 and here’s the gist: you soak the cells & DNA in lots of calcium chloride (CaCl₂). The positively-charged Ca²⁺ neutralizes the negatively-charged DNA & membrane surfaces, allowing the DNA to stick to the membrane near “tunnels.” Initially, these tunnels are too small for the DNA to get through, but when you heat the cells, the tunnels open up & the DNA rushes in.
Works great for these bacteria, but to make them “chemically competent” you have to nearly kill them and then the heat shock probably isn’t much fun for them either. Bacteria are pretty hardy so they can take it, but other types of cells probably wouldn’t make it! So another method that you can use with bacteria *and* with other types of cells is ELECTROPORATION. This is another way to open up tunnels into cells and get DNA to go into them.
Eelectroporation is aka electropermeabilization, and as these names suggest, it uses electricity to generate temporary pores that make it “permeable,” permitting DNA to enter a cell. You can do the electroporation in a cuvette – similar to those you might have used in a spectrophotometer to check absorption in a Bradford assay or something (more here: http://bit.ly/2CHk9Xv ) and you give it an electrical pulse. You don’t want to fry the cells, just make small temporary holes, so you only use an electrical pulse that lasts for a few microseconds (millionth of seconds) to a millisecond (thousandth of a second). The pulse disrupts the membrane(s)’ phospholipid bilayer, causing the phospholipids to “shift,” opening up pores.
And it’s not just phospholipids that are shifting. Ions (charged molecules) are too. Electricity involves the movement of charged particles. If you separate 2 opposite charges, it’s kinda like taking a bowling ball to the top of a cliff and holding it over the edge. There’s a lot of “potential” for electricity (which we can measure in volts (V)).
When you turn on the power, it’s like letting go of the bowling ball – the negatively-charged molecules rush towards the + end (and vice versa). This creates an electrical gradient (think of the one used in agarose gel electrophoresis) whereby one side of the cuvette is + charged and the other side is negatively charged.
Just like negatively-charged DNA moves towards the + charged end of an agarose gel because you set up an electrical gradient, the DNA will move towards the + charged side of the cuvette, and, if it happens to be next to an open pore it will move into the cell.
The conditions have to be optimized for different cell types and you’re likely going to kill a lot of cells in the process. But if you do it right, you can end up with much greater transformation efficiency (more cells taking in the DNA) than with chemically competent cells.
Once the DNA’s in there, when using bacteria you have to let the cells recover in antibiotic-free media, giving them time to build up resistance before you add the antibiotic you’re using to kill off cells that haven’t taken in any plasmid, similarly to as if you had used chemically competent cells.
A nice thing about electroporation is that it can be used for “any” type of cell. Unlike competent cells, which require pretreatment, the “pretreatment” for electroporation is simply washing the salt off. In chemically competent cells, you NEED the salt – specifically the high concentration of Ca²⁺, which neutralizes the negative charges of the membrane surface and the DNA so they can stick together. BUT in electroporation, you don’t want the DNA’s negative charge hidden because that negative charge helps provide a motivating force for the DNA to enter the cell. Also, if you have too much salt, something called ARCING can occur, where the charge just whizzes around the cells (path of minimal resistance), rather than through them.
But what if your cells are on a plate or something, so you don’t want to/can’t electroporate?! Another option is to use CATIONIC CARRIER MOLECULES, such as PolyEthyleneImine (PEI), that help cells “swallow” the DNA. This is similar in concept to how we use calcium chloride to make chemically competent bacterial cells. That works great for bacteria, but you have to modify the cells and really stress them out. To get DNA into more “complex” cells, you can take advantage of their more “complex” machinery.
This machinery includes “swallowing organs” that allow for ENDOCYTOSIS -> it’s kinda like a biochemical sinkhole -> basically the membrane sucks in a part of itself (including anything bound to it) and pinches it off (inside the cell) so that, although whatever was bound to it is now inside the cell, it’s surrounded by a membrane, like a little membrane-bound packet
If we get DNA to bind to the membrane, it can hitch a ride inside. So we need to get it to bind the membrane – so we’re back to that idea of finding a good cation.
Enter PEI -> Polyethylenimine (PEI) is a cationic polymer (chain of similar repeating units). PEI’s repeating units are amine (nitrogen-hydrogen) groups with ethyl (CH₂CH₂) linkers, and they can branch off of each other tree-like. DNA’s attracted to it, so they snuggle up together, forming a nice compact blob called a POLYPLEX.
This polyplex has a net positive charge, and is attracted to the negatively-charged membrane surface. So it sticks there. Then the cell membrane “swallows” it through endocytosis.
So, now you have the DNA in the cell, but it’s trapped in this membrane-bound packet called an ENDOSOME (kinda like the cells are “cheeking” a pill). You still need to get it out of there and into the general cell interior (cytoplasm) -> you need it to undergo ENDOSOMAL ESCAPE
Thankfully, the PEI’s + charge comes to the rescue again -> inside the cell there’s lots of CHLORIDE ions (Cl⁻) which are anions (negatively charged). They rush into the endosome to even out that charge imbalance. They might even out the charge, but they “uneven-out” the amounts of “stuff” inside and outside of the endosome compared to the amount of water.
This creates something called OSMOTIC PRESSURE – more on osmotic pressure here -> http://bit.ly/2UjNqwN
Water rushes in to “balance things out” but the membrane can’t handle it -> its kinda like filling a water balloon with a water hose -> too much rushes in and the endosomal “balloon” pops, releasing the DNA into the cytoplasm
Other cationic polymers include DEAE-dextran, Polybrene, polylysine. There are other types of carrier molecules too, and companies offer a variety of “transfection reagents” made up of proprietary mixes of such molecules, often optimized for different cell types). The important things they need to be able to do are 1) get the cell to swallow the DNA and 2) get it to release the DNA once inside.
side note: PEI also comes in handy for a sort of opposite reason during protein purification. One of the first steps in purifying a protein is to break the cells open (lyse them) and separate the nonsoluble stuff (like membrane pieces) from the soluble stuff. Normally DNA is soluble, but if you add PEI, you can get the DNA to precipitate (become insoluble), so you can separate it from your soluble proteins. It’s cool – you add PEI dropwise to your lysate and it gets whiter and thicker as polyplexes of DNA:PEI start forming and precipitating. Then, when you spin the lysate, the DNA ends up pelleting with the membrane so you’re left with a less “gooey” supernatant (the liquid part) to work with. But you have to be careful – If your protein is acidic (negatively-charged) at the pH you’re working with, it could also bind the PEI and get precipitated. You can use a high salt concentration (which you often use in lysis anyway to help rupture the membranes) – this way, the protein will bind the salt instead of the PEI. But the DNA is more negatively-charged and positive-charge-hungry, so it’ll still get wrapped up in the PEI and precipitate.
In addition to and/or instead of cationic polymers, many of the transfection reagents contain cationic lipids. Kinda like the phospholipids in our cell membranes – they have a hydrophobic tail and a charged head, but these have a positively-charged head, so they can glob onto the negatively-charged DNA to help sneak them in. Analogously to how “polyplex” is used to describe the cationic polymer/nucleic acid blob, “lipoplex” can be used to describe the cationic lipid/nucleic acid blob. Another terminology note – the use of lipids for transfection is sometimes referred to as “lipofection.”
One of the names you might hear used a lot in labs is “lipofectamine.” As the “amine” in the name suggests, this transfection formula contains lipids with head groups that contain amine groups. The 2 used in lipofectamine are N-(I-(2,3,-dioleyloxy)propyl)-N,N,N,-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE). DOTMA is a cationic lipid, but DOPE? Nope! It’s neutral. This “helper lipid” helps to get the lipoplex membrane to merge with the cell membrane (either at the surface or inside).
I’m still not clear on the exact differences, but, when mixed with other stuff and nucleic acid, cationic lipids can self-assemble into Lipid NanoParticles (LNPs) that coat and protect the DNA/RNA, help it get taken into the cell, and then help it release it inside. One way it can do this is by promoting endosomal escape by merging its lipids with the endosomal lipids to disturb the endosome and let out the nucleic acids. You can use this to deliver nucleic acids into people, and it is being used by Modern for their SARS-CoV-2 mRNA vaccine, which delivers mRNA for making the virus’ Spike protein so that the body can learn to recognize it as foreign and make antibodies against it, etc.
Speaking of getting nucleic acids into people, you can apparently also use electroporation on people, through a sort of electrode patch but I’m not entirely sure of the specifics and logistics.
Those were all non-viral transfection methods, but there are also “viral vector” delivery systems, where you can adapt a harmless virus to serve as a carrier (vector) for genes you want to put in. The virus can then infect cells and use its virus-y tricks to get the DNA or RNA in there without the cell destroying it. One virus that’s used for this is adenovirus, which is a sort of cold virus, and you can learn more about such viral vector delivery systems here: https://bit.ly/adenoviralvectors
more on chemically-competent cells: http://bit.ly/2Jj7L47
more on agarose gel electrophoresis: http://bit.ly/2SDKE8I
more on molecular cloning: http://bit.ly/2SkSG58
#365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0