07/28/2025
Want to know more about the GFP gene?
I thought so…
GFP stands for Green Fluorescent Protein. It is a small, barrel-shaped protein with a small section called a chromophore. The chromophore absorbs short-wave light (from the ultraviolet and blue part of the spectrum) and re-emits the energy that was absorbed as green light. So GFP axolotls glow bright green under blue, violet, or ultraviolet light.
GFP was first identified in 1962 by Osamu Shimomura, an Organic Chemist and Marine Biologist. Originally, it was identified in a species of jellyfish, Aequorea victoria. Exactly why the jellyfish needs GFP is unconfirmed, but I can hypothesize a likely function for it: My guess is that it is not just the green glow that is of benefit to the jellyfish (and other organisms that naturally have GFP and similar fluorescent proteins) - It is also the fact that GFP absorbs UV light. I’ll get back to that in a bit…
In 1991 Douglas Prasher first sequenced the gene that produces GFP, and suggested that GFP could be used as a tracer molecule in living cells: If you could put the gene into a cell in a developing embryo, you would be able to see all of the cells, tissues, and organs that cell had formed in the organism by just shining a UV flashlight on it! If you attach the GFP gene to any other gene you would be able to see exactly where and when an organism used that gene in its body. He should have received the Nobel Prize for that work - but didn't.
Within just a few months of the publication of Prasher’s work, researchers began plugging the GFP gene into all kinds of research organisms, starting with bacteria, then algae, various plants, roundworms, etc. Within a few years there were even green-fluorescent cats, mice, and monkeys.
The first GFP axolotl was produced in 2005 by Lidia Sobkow and others. Their research was published in March of 2006. The researchers were trying to study the origin of the cells that form certain tissues and the contribution of blood cells to regenerating tissues. They inserted a piece of DNA called a plasmid containing the GFP gene into axolotl zygotes (the first cells produced when s***m and egg join) by directly injecting them.
Some of the resulting embryos became mosaics with some GFP tissue here and there, but some of them glowed all over - including the germ cells that form the te**es and ovaries. That means they could pass the GFP gene on to their offspring! These axolotls became the GFP marvels many of us have in our aquaria today.
So why have so many organisms been genetically modified with GFP?
There are a LOT of ways GFP can be used in Biology! Here are a few:
(1) Tracking Cell Fate and Development:
If you transfer a cell or cells from an embryo with GFP into an embryo without GFP, you can actually watch as the GFP cells form tissues and organs. You can see exactly where the cells migrate in the body and what parts of the body they ultimately become.
That’s what I was doing with my students when we produced the first “firefly” axolotls many years ago. We could see how the migration of pigment cells in leucistic (white) axolotls from one embryo were affected by cell-signalling proteins from dark (non-white) cells transferred to them - and vice-versa.
You can even label cancer cells with GFP and see how they metastasize, forming tumors in other parts of the body.
(2) Protein Localization:
You can connect the GFP gene to any other protein-encoding gene. As a result, when the organism produces that particular protein it fluoresces, allowing scientists to know exactly where the protein is being produced, and whether and how the protein is moved around in the organism’s body.
(3) Gene Expression Monitoring and Biosensors:
GFP can be used as a reporter gene. When attached to parts of the DNA called promoter genes, the glow of the GFP fluorescence indicates when and where that promoter is active, allowing scientists to study how promoter genes regulate the expression of other genes.
Once you understand how the promoters work, you can deliberately connect GFP to a specific promoter so you can see when a particular gene is activated. For example, you could attach it to a gene that is only activated in response to a certain environmental toxin, turning a plant or bacterium into a fluorescent poison detector.
You could attach GFP to the W chromosome of a chicken, causing only female embryos to glow. By shining a UV light through an egg, you could tell which ones will produce hens and which will produce roosters, or instantly identify hens when they hatch. If you don’t know why that would be awesome, look up the horror show that is the egg-production industry. This would be an easy gene edit using CRISPR, btw.
You could also do that for axolotls… Gee… I wonder if anyone is doing that even now?...
Since the discovery of GFP we have modified the gene to produce fluorescent proteins that produce other colors. We now have a whole rainbow of fluorescent color genes to use in research. This allows us to label multiple cells in a single organism. You may have seen, for example, the recently published map of the neurons in a small piece of a human brain with all of the individual neurons glowing a different color! By doing things like that we can begin to understand how neurons connect and interact to construct a living brain.
If you’re wondering why we don’t, therefore, have red, blue, orange, yellow, etc. fluorescent axolotls in our tanks now: It is because most of those other fluorescent proteins generally suck in an aquarium. They don’t fluoresce nearly as brightly, and they all absorb different wavelengths of light. GFP absorbs short-wave light - mostly in the high-energy UV range that our eyes can’t see at all - then returns the energy as low-energy green light that we see very well. That makes their glow very bright and easy to see. The other colors of fluorescent proteins that are based on GFP just don’t work that way.
There are, for example, quite a few Red Fluorescent Protein (RFP) axolotls in the pet trade in the USA. I know that because I put a lot of them out there - and didn’t bother to tell anybody.
Edit: There were already unidentified RFPs in the pet trade before I did this (in 2016), so it didn't come from me! I found my first (known) RFP a few years before in a random clutch that was given to me. That should tell you just how little the RFP gene affects phenotype.
The RFP axolotls don’t glow under UV, they glow under cyan light. Our eyes are very sensitive to cyan light. You have to shine a bright cyan light on the RFP axolotl to make it glow, and then it glows VERY faintly red - which you can’t see because of all the cyan. You need glasses with a filter to remove the cyan light so you can even see the red glow. Don’t misunderstand: I think RFP axolotls are very cool, but they just don’t have the literal flash of the GFPs.
Our GFP axolotls can glow very brightly when exposed to bright UV light - especially 365nm UV.
They don’t like it.
Keep in mind that (a) they can see UV light as easily as we see red, green, or blue, (b) they have no eyelids, and (c) the GFP is expressed in every cell of their bodies - including their eyes! This means that a UV light is not just annoying to them - It is intense and inescapable. While you will not harm your axolotl by occasionally zapping them with a UV flashlight to see the GFP, or to check for the blue fluorescence of an axanthic or hypomelanistic, it should be done briefly and infrequently. Frequent exposure to bright UV light is very stressful, ultimately causing appetite loss, hiding behaviour, and other issues.
Ultraviolet light is ionizing radiation. That means that it damages organic molecules like those that make up living cells.
You (and all other mammals, reptiles, and birds) don’t have living skin on the outside. The cells of your epidermis are actually dead. When you look at another person, everything you’re looking at is dead, in fact: Hair, nails, corneas, skin - It’s all dead, keratinized cells, stacking in layers like shingles on a roof. That outer layer of dead cells seals your skin like plastic wrap to reduce water loss, infection, and injury. The dead cells are also loaded with dark melanin, which absorbs UV light so that it can’t reach the living cells underneath. Your skin produces extra melanin in response to UV exposure, which is why we tan - but the process is slow and takes a while to produce enough melanin, which is why we also burn. We “burn” in sunlight because the UV light destroys proteins and DNA in living cells, killing those cells pretty quickly.
It’s worse for axolotls and other organisms (all fish and amphibians) that have living exterior skin. They don’t produce keratin the way we do, and they don’t have a protective, shingled layer of melanin-absorbing, dead cells as an outer covering. Their skin is alive and exposed, like an open wound. They have to avoid exposure to UV light as much as possible, which is why they generally avoid bright light - even when they don’t have GFP.
Fun fact: ALL axolotls are sensitive to UV light ALL OVER THEIR BODIES! They have photosensitive cells scattered throughout their skin that are connected to their sensory nervous system. They can “feel” that UV light in much the same way we can feel the warm infrared light of the sun or a campfire. Even eyeless axolotls will respond with some annoyance when exposed to UV light. As explained earlier, this is to protect them from the damaging effects of UV light.
So why do the Aequorea jellyfish have GFP in the first place? The following includes some speculation, and doesn’t say much about axolotls, so I won’t mind if you stop reading here.
Most animals have eyes that are very sensitive to short-wave light. Most animals other than mammals have photoreceptors in their eyes that detect ultraviolet light. That means that many fish, amphibians, reptiles, and birds look at a rainbow and see not just ROYGBIV, as we humans do. Their rainbow is much wider than ours, extending quite a bit past the violet end. Many of those animals also have other color-sensing receptors that allow them to distinguish colors between those we see, and some, like some species of snakes, see well into the infrared at the other end of the rainbow, too.
Mammals seem to have lost almost all of their ability to distinguish colors around 65 million years ago or so, probably because our ancestors then were either burrowers and/or nocturnal animals that spent most of their time in the dark. Not much need for color when there isn’t anything to see. Mammals did keep the photoreceptors that are sensitive to blue and red, though. Why? Because blue light has a lot of energy per photon, it is visible even in dim light. You can see blue in moonlight - but not green or red. That’s why kids tend to use a lot of blue crayon when drawing night scenes. Red light is weak, but is great for seeing contrast against the green and blue background of plants and water in daylight. There’s not much need to see green when your world is mostly a blur of green leaves and water.
Many predatory fish, amphibians, and reptiles, and almost all of the nocturnal predators, have lost their long-wave color vision. Again, there is no reason for a predator to be able to tell green from red or yellow. They don’t eat leaves, strawberries, or bananas (ask your cat). They DO, however, need sharp vision in dim light. Their eyes are chock-full of “rod” cells that detect a wide range of light waves from infrared to UV (so can’t distinguish color), and UV and blue-light sensors that can help see detail in very dim light.
If you’re a relatively defenseless, easy-to-catch animal like a small jellyfish, it helps to be invisible to things that want to eat you. One way to do that is to have markings on your skin that match your surroundings - but that is useless in the open ocean where your surroundings are just an endless sea of blue water. You could be blue, but you would have to be just the right shade of blue so that you blend in from whatever angle the predator is approaching from. From the top you need to be dark, and from the bottom very light.
A lot of aquatic and marine animals actually do use that strategy. A bluefin tuna, for example, is literally dark blue on the back, light blue on the sides, and white on the belly. The sun shining from above lights up the dark back, while the light belly is in shadow. This is called “countershading”, and is very common for animals that live in brightly lit, exposed places. Even deer have it! Countershading makes the fish harder to see from any angle unless you are pretty close to it. Fish will even angle their bodies , presenting their back to the sunlight, to maximize the effect of countershading. You can watch the fish in your aquarium do that if you put a bright light on one end of the tank.
Small jellyfish use another strategy: Most are simply clear. Light goes right through them… er… mostly. Unfortunately for them, the shorter, high-energy wavelengths of light, including indigo-blue, violet, and ultraviolet, don’t pass through living tissue as easily as red, yellow, or green light. This means that a predator with good UV vision looking up from below at a “transparent” jellyfish would see a dark body silhouetted against the sunlit water overhead. Even countershading won’t help in that situation.
This is where the GFP comes in: Those dim-light predators see UV and blue light very well. If you can absorb the UV light, it doesn’t reflect off your skin. It works like the radar-absorbing skin of a stealth jet. UV-sensitive predators looking at an Aequorea jellyfish from above see only the dark depths of the dark sea below them.
I suspect that this is the reason many other animals fluoresce green or red, like scorpions. They absorb the UV light their predators (and prey) can see, and re-emit the energy as red and green that their predators can’t see.
That still doesn’t prevent the jellyfish from making a dark shadow against the sky, though. What DOES make the shadow disappear is to glow green! Remember that most deep-water and nocturnal predators can distinguish the colors blue through ultraviolet - but not red and green. Even though they also have a lot of rod cells that can see in dim light, those rods can’t distinguish one color of light from another. They see only degrees of light and dark, like an old black-and-white movie - just as you do in moonlight. The green glow from the jellyfish lights up their shadow when seen from below, making it brighter against the lighter, sunlit water above them. To a predator that can’t distinguish green from blue sky or water, the jellyfish disappears from below.
But wait, you say: Green-glowing jellyfish would stand out like searchlights to a predator when seen from above - and you’d be correct. Remember, though, that GFP only glows green when it absorbs UV light. That means the brightness of the glow is tied to how close the jellyfish is to the surface and how much sunlight is coming in from above. They glow brightly in bright sunlight, and not at all in the dark. In deep water they are dark, absorbing all of the UV light that hits them, but glowing green too faintly to be seen from any significant distance by animals that can’t see the color green. The closer they get to the brightly-lit surface of the sea, the more brightly they glow green, which precisely cancels out their shadow as seen from below!
Those of you with GFP axolotls can see this effect very clearly by moving the axolotl near a sunlit window. Don’t do it for long, though: As I said earlier, axolotls don’t like UV light at all, and GFP axolotls REALLY don’t like it.
