GFP Axolotls Video

[gr33neyes]

26th November 2003, 16:39 #10 jennifer

Posts: n/a
Gallery Images:
Comments:

There are some fish with dyes added to make them brightly colored. The glo-fish zebra fish are like this, but the dye is genetic, not pumped in.

There are also gene-spliced animals that contain green fluorescent protein (GFP). GFP is the protein that makes bacteria on the surface of the ocean glow at night. It really does produce it's own light (not just being bright or reflective). Glowing zebrafish and Xenopus already exist in labs. And I wouldn't be surprised if glowing axolotls are produced some day.

...............found this in a search. looks like her prediction was right.....................................

 

[Julie1002]

i think its kind o creepy. Wouldn't the constant "glowing" disrupt the natural habits of axies as they are nocturnal creatures?

 

[Saspotato]

They only glow when UV lights are pointed at them so I don't think it would be an issue. Correct me if I am wrong...

 

[gr33neyes]

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

This may be of interest.

 

[ben_tajer]

I don't know if I agree with GM or not its such a hard debate, first of all you are taking away the survival of the fittest from natures hands which can have unseen conquences, but thinking about what GM can offer, starving families could have a GM meat or vegatables which has a great nutritional value to help them survive.

Random fact of the day: Cat pee glows under black light.

Well, by seperating out your larvae and rearing them without predators aren't you taking away survival of the fittest? By not keeping your adult pets with predators aren't you also preventing a "natural" process? People have been modifying and interfering with nature for thousands of years, I don't see why now, when we can suddenly do it faster and more efficiently, it has to become some sort of ethical debate. Glowing axolotls, and other glowing animals are not only a good proof of concept, but may also do greater things, like displace the demand for wild caught animals. And what's more, with glow in the dark animals, incidents like the "teenage mutant Ninja turtle incident" (the popularity of teenage mutant ninja turtles caused children around the world to buy red eared terrapins by the millions, when the terrapins out grew their enclosures they were often released, becoming established in parts of europe and having dramatic environmental consequences), because there would be next to no chance of the glowing animals becoming established in the wild. GM could not only give us more crops but for example we could genetically modify algae to produce bio fuels or sequester carbon more efficiently than any plant in the wild, we could create viruses targeting pest species and invasive species (we've already done this to a small degree), we could create species that would facilitate deep space exploration and colonization, and going into more controversial territory, we could eliminate Sickle cell, Cystic fibrosis, maybe even some forms of depression and cancer. The possibilities are endless, and if it weren't for this wealth of potential, I wouldn't want to be a biologist.

People often argue that GM populations would be more vulnerable to disease, but I don't see why this would have to be the case. Sure if every GM crop were a clone of the last this would be true, but we can make it so that GM crops are genetically diverse, we can also possibly genetically engineer new strains in the face of a disease faster than the plants would adapt naturally. Sure there's the odd mishap, such as the issue with brazil nuts and allergies, but these are often sensationalized. Journalists are looking for genetic frankensteins because it makes for and interesting story and sells more articles. The truth is, as Matt Ridley states in his book The Human Genome, those Brazil nuts had the potential to benefit a lot more people than they would harm, but the media jumped on it and smothered it.

Some people think genetics will result in eugenics, nonsense, nazism causes eugenics. I don't see why it's such a hard debate, please enlighten me. I see GM playing an essential role in a brighter future.

 

[Abrahm]

I don't see why it's such a hard debate, please enlighten me. I see GM playing an essential role in a brighter future.

I'm pro-genetic modification for the most part. Hell, I've already genetically modified yeast and bacteria to produce frog proteins. I agree that it will make a brighter future but there are also many abuses of the science and technology. I don't think you can easily say that this is not a hard debate. There are a multitude of aspects that make this potentially difficult.

What about things like terminator genes that make farmers buy their crops every single year instead of using last years seeds? What about scientists and companies patenting genes and charging huge amounts of money for their new anti-pest corn?

Some people think genetics will result in eugenics, nonsense, nazism causes eugenics.

Whoa, that's some horrible logic there. National socialism didn't cause eugenics, they just used it to further their own horrible ends. Eugenics was around before that and is still around.

It's an easy thought to have that if genetic testing is available and one can find out if a fetus has cystic fibrosis and choose an abortion that they might do the same for something aesthetic. There are a lot of details to work out with genetically modified organisms, genetic testing and other aspects of biotechnology.

because there would be next to no chance of the glowing animals becoming established in the wild.

Why? There's no reason that these animals couldn't survive in an appropriate climate. They won't be subjected to UVA radiation at night so they won't fluoresce. Even in most lakes or ponds I doubt they would get enough UVA to fluoresce during the day.

 

[Levo]

Well, by seperating out your larvae and rearing them without predators aren't you taking away survival of the fittest? By not keeping your adult pets with predators aren't you also preventing a "natural" process? People have been modifying and interfering with nature for thousands of years, I don't see why now, when we can suddenly do it faster and more efficiently, it has to become some sort of ethical debate. Glowing axolotls, and other glowing animals are not only a good proof of concept, but may also do greater things, like displace the demand for wild caught animals. And what's more, with glow in the dark animals, incidents like the "teenage mutant Ninja turtle incident" (the popularity of teenage mutant ninja turtles caused children around the world to buy red eared terrapins by the millions, when the terrapins out grew their enclosures they were often released, becoming established in parts of europe and having dramatic environmental consequences), because there would be next to no chance of the glowing animals becoming established in the wild. GM could not only give us more crops but for example we could genetically modify algae to produce bio fuels or sequester carbon more efficiently than any plant in the wild, we could create viruses targeting pest species and invasive species (we've already done this to a small degree), we could create species that would facilitate deep space exploration and colonization, and going into more controversial territory, we could eliminate Sickle cell, Cystic fibrosis, maybe even some forms of depression and cancer. The possibilities are endless, and if it weren't for this wealth of potential, I wouldn't want to be a biologist.

People often argue that GM populations would be more vulnerable to disease, but I don't see why this would have to be the case. Sure if every GM crop were a clone of the last this would be true, but we can make it so that GM crops are genetically diverse, we can also possibly genetically engineer new strains in the face of a disease faster than the plants would adapt naturally. Sure there's the odd mishap, such as the issue with brazil nuts and allergies, but these are often sensationalized. Journalists are looking for genetic frankensteins because it makes for and interesting story and sells more articles. The truth is, as Matt Ridley states in his book The Human Genome, those Brazil nuts had the potential to benefit a lot more people than they would harm, but the media jumped on it and smothered it.

Some people think genetics will result in eugenics, nonsense, nazism causes eugenics. I don't see why it's such a hard debate, please enlighten me. I see GM playing an essential role in a brighter future.

That is a very strong argument and there is nothing really I can disagree with in the whole matter, it is true and the survival of the Human race all together has relied on the ability to cheat survival of the fittest, the fact that I am able to sit at this computer wearing a pair of glasses just shows how I have cheated survival of the fitesest left to my own devices of nature I would be long gone not being able to see. I do like the idea aswell of these GFP axolotl's, I don't know if this has been anwsered but if the pair did breed would there offspring already have the GFP inside them at birth? Because if you were able to breed them like that I think it would be a great idea from an aesthetic point of view, as long as the well being of the animal isn't suffering.

There is always going to be some concern and it is worth looking at those as well, the fact that nature has a good way of being one step ahead of us. We try and change something, nature will try and find another way to control it.

 

[Kal El]

I must admit that this is a very intriguing topic, though I have never heard of GM axolotls for bioluminescence, it does pose some complications.

Perhaps a background in luminescence would be useful: Fluorescence (in the case) is a form of luminescence, and seldom occurs because there aren't many molecules that fluoresce. Within the molecule itself, are molecular orbitals and when an excitation radiation, UV light, is directed on the molecule, it causes an electronic excitation. That is, an electron from the lowest molecular orbital (the ground state) of the molecule is promoted to an excited singlet state (a higher in energy molecular orbital). The electron will remain in this excited singlet state for approximately 10E-9 seconds to 10E-7 seconds before it returns to the ground state again. Upon its return to the ground state, a photon of light is emiited from the molecule. This photon of light is what we see, and is unfortunately coined as "glowing in the dark", when speaking colloquially.

NB. Singlet states can only result in fluorescence. Triplet states correspond to phosphorescence (another form of luminiscence but is less common because it must be conducted with miscelles and at -77 Kelvin = -350.15 degree celcius- the temperature of liquid nitrogen)

What must be noted is that during the transition from the ground state to the excited singlet state, there is a change in molecule's structure for a brief period of time- approximately 10E-9 seconds to 10E-7 seconds (as mentioned above). Thus, this poses a problem in itself. During that brief perioid biological/chemical reactions can occur that would inevitably effect the axolotl's anatomy. The first biological molecules to be harmed would most likely be enzymes.

Another thing to consider is that, the excitation radiation is UV. This form of light can be extremely damaging to the epidermis of amphibians because they lack the ability to protect themselves from any source of light.

I'm not against GM but obviously some aspects need to be taken into account before conducting such practical experiements. I'm also aware that's a hard shot to call because inferences need to made before observations can be noted. But such is a life of a scientist- optimistic to the end.

Jay.

 

[ben_tajer]

Abrahm: You do make a good point about them not glowing in the wild, but would these animals necessarily be prepared for existence in the wild? The ones in the video appear to be albino, and the production of a useless florescent protein would burden the animal to a certain degree, possibly reducing the animal's competitiveness in a situation where resources are limited. I was negligent to ignore the risks associated with the animals spreading to the wild, but in a domesticated modified form, I think it will often be the case that the animals are less likely to become invasive than their wild adapted counterparts.

I have to say that I agree with you on the patents and the terminator genes, but I don't really think that these can be used as an argument against GM. This is just bad practice, and as new issues arise, they will have to be legislated for appropriately. Expensive gene patents aren't really helping scientific progress, because they make research more exclusive, more expensive, and less cooperative.

You're right, Eugenics was an issue before the Nazis came to power and the abortion of children due to conditions like down syndrome and cystic fibrosis, is essentially eugenics. While this brings up its own ethical arguments, I was thinking of the Nazi extermination and forced sterilization of fully conscious humans on racist grounds, which though maybe inspired by genetics, is by no means a result of genetic research or technology. Neither are the conclusions about race and affluence suggested by National Socialism and social darwinism respectively, ones reached through the pursuit of genetic science.


Kal El:

I wondered about that too, I never thought mixing amphibians and black lights would be a good idea (I know it's standard practice with some reptiles) as they have thin skin and normally live in dark or aquatic environments. Also I can imagine the heat produced is problematic with animals that are ideally kept below room temperature.

 

[Jake]

That is a very strong argument and there is nothing really I can disagree with in the whole matter, it is true and the survival of the Human race all together has relied on the ability to cheat survival of the fittest, the fact that I am able to sit at this computer wearing a pair of glasses just shows how I have cheated survival of the fitesest left to my own devices of nature I would be long gone not being able to see. I do like the idea aswell of these GFP axolotl's, I don't know if this has been anwsered but if the pair did breed would there offspring already have the GFP inside them at birth? Because if you were able to breed them like that I think it would be a great idea from an aesthetic point of view, as long as the well being of the animal isn't suffering.

There is always going to be some concern and it is worth looking at those as well, the fact that nature has a good way of being one step ahead of us. We try and change something, nature will try and find another way to control it.

Axolotls aren't 'born' like some other animals, the eggs are fertilized internally and then layed. They do have the gfp gene when they begin to develop, but don't start to glow until tail-bud stage. The guy who bred the axolotls in the video started with one white gfp male, and now he's bred enough to share with plenty of people.

 

[rich]

When it comes to GM im on the fence, on the one hand it can help millions of lives, like getting a cow to produce insulin in her milk. yet when it come to changing animals or crops for there asphetic trates then i am against it, as it provieds no help to the many. (Even tho most labs will tend to argue that doing these tesets {the GFP axie for example} is for the sientific world to understand how to obtain certain genes, and do the tests and see what produces the best outcome so it can be used to help the world) ... This to me doesnt sit right, as in japan you can obtain these glowing fish ect, and there is also a case of a farmer getting his pigs to glow in the dark so he can see them at night (i forget where this is but studied it at college). As long as these axies are not comercially produced for people to own as pets i have no problems with it, as long as theye are not mis treated (which as i understand there not )

 

[Kal El]

Just out of curosity, does anybody know the lifetime of the excited singlet state for GFP? Because I read in that wikipedia link that GFP has a complex structure that protects it from queching. The reason why I'm asking is because, I want to know whether phosphorescence is possible? Despite the ridiculously low temperature requirements (77K). But if GFP were isolated, would it possible for phosphorescence to occur?

After all, phosphorescence does have a more attractive (dare I say ) luminescence to it because of the characteristic "after-glow".

Jay.

 

[Abrahm]

When it comes to GM im on the fence, on the one hand it can help millions of lives, like getting a cow to produce insulin in her milk. yet when it come to changing animals or crops for there asphetic trates then i am against it, as it provieds no help to the many. (Even tho most labs will tend to argue that doing these tesets {the GFP axie for example} is for the sientific world to understand how to obtain certain genes, and do the tests and see what produces the best outcome so it can be used to help the world) ... This to me doesnt sit right, as in japan you can obtain these glowing fish ect, and there is also a case of a farmer getting his pigs to glow in the dark so he can see them at night (i forget where this is but studied it at college). As long as these axies are not comercially produced for people to own as pets i have no problems with it, as long as theye are not mis treated (which as i understand there not )

The fluorescent pigs, axolotls and frogs are all great scientific tools. Axolotls for regeneration, frogs because they are easy to modify, and pigs because they are physiologically similar. GFP can be used in a variety of ways to help us understand expression and regulation of genes and protein production. In our lab, GFP modified animal cells were used to help determine that our protein of interest appeared to localize in RNA processing areas.

Just out of curosity, does anybody know the lifetime of the excited singlet state for GFP? Because I read in that wikipedia link that GFP has a complex structure that protects it from queching. The reason why I'm asking is because, I want to know whether phosphorescence is possible? Despite the ridiculously low temperature requirements (77K). But if GFP were isolated, would it possible for phosphorescence to occur?

After all, phosphorescence does have a more attractive (dare I say ) luminescence to it because of the characteristic "after-glow".

The singlet state for GFP is on the nanosecond scale. I don't think phosphorescence is possible, but I don't understand the underlying quantum chemistry enough to speculate.

Where did you get this information on phosphorescence only occurring at 77K? I could see certain compounds that only phosphoresce at low temperatures, but there are still compounds that phosphoresce at room temperature. Shirts that glow in the dark after being charged and kids toys are two easily found examples.

 

[ben_tajer]

When it comes to GM im on the fence, on the one hand it can help millions of lives, like getting a cow to produce insulin in her milk. yet when it come to changing animals or crops for there asphetic trates then i am against it, as it provieds no help to the many. (Even tho most labs will tend to argue that doing these tesets {the GFP axie for example} is for the sientific world to understand how to obtain certain genes, and do the tests and see what produces the best outcome so it can be used to help the world) ... This to me doesnt sit right, as in japan you can obtain these glowing fish ect, and there is also a case of a farmer getting his pigs to glow in the dark so he can see them at night (i forget where this is but studied it at college). As long as these axies are not comercially produced for people to own as pets i have no problems with it, as long as theye are not mis treated (which as i understand there not )

What do you mean by "provieds no help to the many"? Science is beneficial for human kind and the environment alike. Animal testing not only helps humans but it also helps us better understand animals and the environment. As far as I'm concerned the controlled use of animals to reveal better means of both human welfare, animal welfare, and environmental management, is not only justified, but a moral obligation.

Not that you mentioned any of these things, but think a lot of this fear of GM comes from either those who have a poor understanding of the use and importance of animals in science, those who have a problem with "playing god" or interfering with nature, and those who are generally cautious about science and new ideas. I don't see what's wrong with meddling with nature (and I by no means advocate reckless meddling with nature and the environment without a thorough understanding), if we can make it more sustainable (currently by not "playing god" we're filling the air with CO2, chopping down forests, and over fishing - causing "animal suffering" on a scale that scientific testing or factory farming can't even approach). Also why does it have to by referred to as "playing god"? Couldn't we be god's instrument? The bible does mention that it will one day be "on earth as it is in heaven", who says science won't lead the way?

 

[Kal El]

The singlet state for GFP is on the nanosecond scale. I don't think phosphorescence is possible, but I don't understand the underlying quantum chemistry enough to speculate.

Where did you get this information on phosphorescence only occurring at 77K? I could see certain compounds that only phosphoresce at low temperatures, but there are still compounds that phosphoresce at room temperature. Shirts that glow in the dark after being charged and kids toys are two easily found examples.

First of all it's always better to explain things with a diagram So, here is one of the Jablonski's diagram:

http://www.photobiology.info/graphics/photochem13.gif

I know that the lifetime for an excited singlet state (S*/S1) is in between 10E-9s to 10E-7s but I wanted to know the the specific lifetime for GFP. See, as the lifetime of S* increases, the more likely the chance of intersystem crossing (ISC). ISC itself has a lifetime of approximately 10E-8s to 10E-7s; it has a similar lifetime to S*, thus ISC will compete with fluorescence. If ISC is favoured and the excited species enters the excited triplet state (T*/T1- more likely T1 than T2 because there is more vibrational coupling), it can either:

- Phosphoresce
- Internally convert (radiationless) to the lowest vibrational level in the ground state.
- Not phosphoresce at all due to collisional deactivation
- Self-absorb

However, in order for phosphorescence to occur it must be conducted under low temperature of 77K because this is the temperature for liquid nitrogen. The principal effect of temperature is on the rigidity of the solvent, and not the spectroscopy of the solute. Abrahm, the reason why you can observe glow in the dark shirts and toys is because species to excited have been encased in organised media/micelles. Phosphorescence is almost never observed at room temp. However, by placing the species on a solid substrate will result in analytically useful room temp. phosphorescence (RTP). RTP showed that the use of organised media such as surfactant micelles or cyclodextrin (CD) cavitives, will modfiy the species to a rigid state, where fluorescence intensity is enhanced and phosphorescence can be observed at room temp. Organised media also protect the species from quenchers such as oxygen, which will quench T*.

So really I just want to know the exact lifetime of S* for GFP.

Jay.

 

[Abrahm]

However, in order for phosphorescence to occur it must be conducted under low temperature of 77K because this is the temperature for liquid nitrogen.

From the paper "The mechanism of long phosphorescence of SrAl2−xBxO4 (0<x<0.2) and Sr4Al14−xBxO25 (0.1<x<0.4) co-doped with Eu2+ and Dy3+" I give you this quote

Green-emitting SrAl2O4, co-doped with Dy3+ and Eu2+ ions, is regarded as a useful phosphor [1] that has very good brightness, long persistent phosphorescence and greater chemical stability than the traditional ZnS: Cu, Co. Since it is not radioactive, this material can be safely used as the phosphorescent pigment for luminous watches and clocks and for “cold lighting” that emits no infrared radiation.

Notice the room temperature examples given such as watches and clocks. Phosphorescence occurs at room temperature and is frequently used in industry without the specific set of experimental parameters that you have listed. You can even find many companies selling strontium aluminate compounds, like this one. Why does the temperature of liquid nitrogen matter? Certainly some compounds will only phosphoresce under those conditions but to say that all phosphors function under those conditions is wrong.

The principal effect of temperature is on the rigidity of the solvent, and not the spectroscopy of the solute.

What? Temperature is the motion of particles. If you mean that with reduced temperature comes reduced entropy resulting in crystalline structures (i.e. solids) and doesn't effect the way the molecule interacts with the electromagnetic spectrum that makes more sense but that is an odd way to phase it.

Abrahm, the reason why you can observe glow in the dark shirts and toys is because species to excited have been encased in organised media/micelles. Phosphorescence is almost never observed at room temp. However, by placing the species on a solid substrate will result in analytically useful room temp. phosphorescence (RTP). RTP showed that the use of organised media such as surfactant micelles or cyclodextrin (CD) cavitives, will modfiy the species to a rigid state, where fluorescence intensity is enhanced and phosphorescence can be observed at room temp. Organised media also protect the species from quenchers such as oxygen, which will quench T*.

I've seen evidence of room temperature phosphorescence and I don't think that such things as yo-yos actually contain complex structures of phosphors trapped in micelles.

So really I just want to know the exact lifetime of S* for GFP.

Try looking this up in pubmed.gov or your University library. They should have a journal archive for you to search through.

 

[oceanblue]

I've found some of the posts in this thread hard to understand and I've spent the last 18 months scouring the literature on bioluminescence!

Green fluorescent protein does what its name suggests. It absorbs energy from a chemical reaction in the jellyfish which would otherwise result in blue light and re-radiates it in the green. The jellyfish actually glows green in the dark, presumably to attract prey (or possibly to identify it as well defended by stings to a predator). The phosphors inside ordinary fluorescent lights show similar properties, converting the ultra violet to longer wavelength visible light.

The axolotl is being used as a living mass of phosphor swimming below an ultraviolet source. It would need a luciferin/luciferase system incorporating into its genome to glow spontaneously.

Luminescence genes have been transposed into tobacco plants, but not as far as I'm aware animals.

Green fluorescence protein will advance our understanding of how animals work, just as many useful techniques such as in vitro fertilization are based on foundations including studies of axolotl embryology. I'm not sure why I somehow feel pet axolotls shouldn't look like this and that leucistic, albino or melanoid and new natural variations should be enough.

Personally I think that if people want to see bioluminescence they should look around and enjoy fireflies, glowworms, dinoflagellates or luminous marine bacteria. I appreciate glowing things have a fascination and am trying to launch in the UK a light based on Photobacterium phosphoreum. I find the glow of the various bags of culture medium which are lying around the house quite relaxing but am content my axolotls remain dark normal and leucistic.

 

[gr33neyes]

:violent::blush::shocked: This post has drifted way over my head. I wonder if you could keep it more simple for us mere mortals who don't have degrees in science and physics and numbers and chemicals that make pretty colours and go bang

 

[ianclick]

Way down here at the bottom of the world we have legislature that prohibits any form of Genetic modification So it would be a moot point in New Zealand.

 

[Kal El]

Yes, perhaps next time it would be good if I didn't attempt to discuss analytical chem. at 2am!

Allow me to try again when my head isn't about to hit the keyboard :lol:

Regarding your first link; I did look over the abstract very briefly but unfortunately I could not open the pdf. file but wtih that sneak peek, I did sort of grasp the concept behind the referenced quote:

"Green-emitting SrAl2O4, co-doped with Dy3+ and Eu2+ ions, is regarded as a useful phosphor [1] that has very good brightness, long persistent phosphorescence and greater chemical stability than the traditional ZnS: Cu, Co."

I presume this would be owing to the heavy atom effect. That is, the mixing (vibrational coupling) of S* and T* is enhanced by spin-orbit coupling (when an electron interacts with the electromagnetic field near the nucleus of a heavy atom, in this case Dy3+ and Eu2+). Enhanced vibrational coupling increases together with the rate constant for ISC (kx). Then if you refer back to what I mentioned earlier, you'll see that as ISC increases, as does the likelihood of phosphorescence ("P" for now- such a long word ). It is true that Cu has been used in the heavy atom effect, in fact it forms a chelate with the analyte/species which does not result in fluorescence at all, but very high "P".

I do believe that 77K plays a role in "P" because it is one of the possible conditions in which "P" will occur. So there are several conditions which will result in "P" will occur:

- Low temp (77K). I will have check this with my professor but I do believe that it has something to do with the rigidity of the solvent in which the analyte/species is in. Possibly because the low temp. discourages entropy (as you mentioned) thus reducing the collisional deactivation (which will occur at room temp. because of the extended lifetime of "P". Approx. 10E-4 to 100 seconds) and interactions with T* quenchers.
- Organised media/micelles
- Heavy atom effect.

Anyway, I think I will check up that last link you gave me and then try Uni. Cheers!

Jay.