How to create a GFP or Bio-luminescent frog?

[Dendro Dave]

First of all hello everyone. My name is Dave and I am heavily involved in several animal related hobbies such as aquaria, naturalistic vivaria, and small mammals like my pet Fennec Fox

 

Anyways I am interested in learning more about the methods used to create transgenic animals. In the hobbies I am involved in we have already seen the introduction of GFP axolotls amphibians and the GLO-fish in the aquarium hobby. I'm interested in learning some of the specifics on how this is done. What transfection methods are easiest and/or best for frog's eggs. What reagents and/or micro injection buffers are needed, and what are some of the procedures. I'm not going to lie, If I found out it was cheap and easy, and the animals were likely to develop into healthy viable adults I might give it a go to satisfy my own intellectual curiosity and to have a unique pet. We are highly concerned with the ethics of line breeding and hybridization in the amphibian hobby especially. And in general I do not favor these practices, not so much on an ethical basis that its wrong in itself but that it can cause practical problems such as not knowing if what you want to buy is actually what you are buying, or messing up the already small gene pool of animals we have to work with. I see transgenic animals who's selling points would be the FX generated as not posing so much risk because there would be little to gain from hiding their genetic origins, and they would be easy to keep separate from other animals if we pressured people to do so...and believe me we are good at pressuring people to stick to our accepted practices in that hobby

 

Anyways I've been having trouble finding, or at least getting access to full papers describing the techniques, specific reagents or injection buffers used.

Just to be clear, I'm super broke, unemployed and generally lack motivation so this isn't on my list of things to do any time soon or likely to ever happen really, at least by me. Nor do the ethical implications escape me, and I still haven't decided if I could do it...would I? But I see no harm in exploring it intellectually if some are willing to help me.

 

So I have been looking into it and it almost looks like this would be possible for someone with a couple thousand dollars to pull off in their kitchen if they could gain access to the chemical materials. Can that be right? Is it as simple as mixing a solution of GFP or lucerfase in a dish with the fertilized eggs and a reagent (which one? lipid based maybe?)and letting them sit at room temp for a while? Or just using a micropipette to directly inject the lucerfase or gfp genes into the egg before it starts dividing? Can this be done by hand? I've got to be missing something here, it can't be as easy as it seems from my admittedly cursory research... is it? Would the effect be likely to be expressed throughout the body, especially the skin? Or would it be a mixed bag of animals showing different levels of expression? Would the effect be temporary or permanent? I assume it would be germ-line and the animals would be capable of breeding normally and passing on the introduced genes at various levels of expression to their offspring right?

 

Thanks in advance for any help exploring this. If anyone wants to post a detailed "how to", or PM me one on the easiest way to do this feel free

 

Sincerely,

Dave

[CharonY]

It would costs far more than a few thousand to accomplish. You would have to first make a transfection construct (i.e. a vector containing GFP or GFP fusion with correct promoters) then transfect and then select for those with successful transfection. Essentially you have to create a mutant that actively expresses GFP (or luciferase for luminescence). While the construction of the vector is relatively trivial (but requires somewhat costly equipment to function efficiently). The actually transfection (i.e. insertion of the vector and hoping for efficient recombination) is a rare event, meaning that you have to screen a lot until you find a cell that got it. Definitely not a very good DIY project.

[Dendro Dave]

It would costs far more than a few thousand to accomplish. You would have to first make a transfection construct (i.e. a vector containing GFP or GFP fusion with correct promoters) then transfect and then select for those with successful transfection. Essentially you have to create a mutant that actively expresses GFP (or luciferase for luminescence). While the construction of the vector is relatively trivial (but requires somewhat costly equipment to function efficiently). The actually transfection (i.e. insertion of the vector and hoping for efficient recombination) is a rare event, meaning that you have to screen a lot until you find a cell that got it. Definitely not a very good DIY project.

 

Ok forgive me because while fairly intelligent and probably more then a layman in most sciences (compared to the average Joe), much of this terminology is not familiar to me. So you may need to dumb it down a bit Let me see if I understand this correctly. What you are saying as far as a transfection construct... That is whatever you are isolating the gfp or lucerfase gene from to have the material to transfect the cell with, correct? As far as evaulating successful transfection for someone in their kitchen I figured they'd basically be stuck with raising the animals and seeing if they glow or not...is that totally naive? I mean the whole point in this particular exercise would be to produce an animal that had the desired visual effect...also personally I'm not to keen on the idea of culling non transfected animals unless it could be determined say with a microscope in the dark and possibly a black light at the early stages of development that they weren't going to express the gene.

 

So I've been checking out this site for Promega, which sells a lot of the necessary genes and whatnot. Would it be possible to do a manual tranfection through micro injection using one of their gene products on this page... http://www.promega.c...asp?subappid=19 or a similar product version of the GFP gene... http://www.promega.c...b320/tb320.html ? Wouldn't a product like that eliminate the need to make your own transfection construct like isolating the gene from the firefly or a bacterial carrier like e. coli? BTW I did stumble on a kit for teachers for under 200 bucks that allows you to insert luminescent bacteria genes into e.coli and another potentially useful kit...http://www.carolina....sortby=ourPicks and http://www.carolina....Picks&refType=4

 

I guess one thing I'm curious about is if I obtained the gfp genes and kits such as those linked to above...what would I be missing that would allow me to do essentially the same thing only with frogs eggs? BTW I realize that achieving labratory efficiency is probably out of the question, but what about a 1 in 100 shot...something like that? LoL or is that laboratory efficiency still

 

Also curious if manual microinjection is outside the realm of possibility for a person at home. It seems the most simple straight forward method if achievable by hand even if the efficiency is poor. Other then steady hands and micropippets Is there a special apparatus I need? I would assume an injection buffer would be necessary at least...correct? If manual micro injection by hand is impossible then what about a method using the raw gene such as products like in the 2 promega links I posted above (especially the gfp gene) and a reagent (lipid based maybe?)mixed in a dish with the freshly fertilized eggs. While writing this reply I was also researching further and I read somewhere I think that lucerfase wasn't enough to get the glow, you need lucerfin also...so perhaps making an animal that has all the genes necessary not just to glow in theory but to actually make itself glow is beyond the reach of a smart guy in his kitchen. What about lux genes or the gfp gene?...Honestly I've just seen lux genes mentioned a time or 2, I'm talking out my ass there I know nothing about them, but I do know that with gfp genes, gfp gene expression is all that is required to elicit the glow under uv lighting, so perhaps that is the more practical avenue. So maybe switch focus and discuss how possible it is for a guy at home to insert the gfp gene into some frogs eggs using methods like microinjection or fertilized eggs bathed in gene/reagent combo solutions. Any thoughts/info there? Much easier...or just as hard?

Edited January 6, 2011 by Dendro Dave

[DavidG]

OK, I have to admit a college student's question got me started down this same path, and I reach very different conclusions from the response given by CharonY.   Its possible that progress in the years since the question was first posted have significantly lowered the difficulty, but I DO see this as an excellent DIY project.

CharonY's comment that the 'construction of the vector is relatively trivial but that the equipment is prohibitively expensive' reminds me of the early days of making a color printout: while the equipment required to produce a color printout was expensive, someone how HAS the right equipment would be open to making a printout on your behalf.  Which had me thinking that anyone WITH the right equipment might be open to the earning a little on the side as a service to those who don't have the equipment.  And with a little searching, I learned that: yep, not only have folks made these services available but that its gone mainstream.  You can order exactly what you need, ready made and ready to go, from places like:

  https://www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorophores/green-fluorescent-protein.html

(I might mention that for me growing up, getting that latest Fisher Scientific catalog was always a highlight of my year!)

With a little more digging around, I found this outstanding publication by Scott that I plan to dive into during the upcoming holiday break:

 'Generation of axolotl hematopoietic chimeras',   http://www.jbmethods.org/jbm/article/view/43/34

Citation: Lopez D, Scott EW. Generation of axolotl hematopoietic chimeras. J Biol Methods 2015;2(1):e12. doi: 10.14440/jbm.2015.43

 

So DaveDendro: did you give up?  Did you make progress?  Did you end up pursuing your line of inquiry?  I hope you did - and would love an update!

  Sincerely, DavidG

[Endy0816]

It is interesting how the cost is coming down, as for a fluorescent frog though evolution actually beat us to the punch.

https://www.nature.com/news/first-fluorescent-frog-found-1.21616

Ironically ran across one Dendro Dave over on dendroboard.com/forum while trying to figure out how importing them might work.

 

The Thought Emporium does a number of 'home' experiments along these lines. It is still almost painful watching the steps needed(and additional expenses necessary) just to have a chance at success.

 

Edited November 13, 2018 by Endy0816

[Olin]

On 1/5/2011 at 10:34 PM, Dendro Dave said:

First of all hello everyone. My name is Dave and I am heavily involved in several animal related hobbies such as aquaria, naturalistic vivaria, and small mammals like my pet Fennec Fox

 

Anyways I am interested in learning more about the methods used to create transgenic animals. In the hobbies I am involved in we have already seen the introduction of GFP axolotls amphibians and the GLO-fish in the aquarium hobby. I'm interested in learning some of the specifics on how this is done. What transfection methods are easiest and/or best for frog's eggs. What reagents and/or micro injection buffers are needed, and what are some of the procedures. I'm not going to lie, If I found out it was cheap and easy, and the animals were likely to develop into healthy viable adults I might give it a go to satisfy my own intellectual curiosity and to have a unique pet. We are highly concerned with the ethics of line breeding and hybridization in the amphibian hobby especially. And in general I do not favor these practices, not so much on an ethical basis that its wrong in itself but that it can cause practical problems such as not knowing if what you want to buy is actually what you are buying, or messing up the already small gene pool of animals we have to work with. I see transgenic animals who's selling points would be the FX generated as not posing so much risk because there would be little to gain from hiding their genetic origins, and they would be easy to keep separate from other animals if we pressured people to do so...and believe me we are good at pressuring people to stick to our accepted practices in that hobby

 

Anyways I've been having trouble finding, or at least getting access to full papers describing the techniques, specific reagents or injection buffers used.

Just to be clear, I'm super broke, unemployed and generally lack motivation so this isn't on my list of things to do any time soon or likely to ever happen really, at least by me. Nor do the ethical implications escape me, and I still haven't decided if I could do it...would I? But I see no harm in exploring it intellectually if some are willing to help me.

 

So I have been looking into it and it almost looks like this would be possible for someone with a couple thousand dollars to pull off in their kitchen if they could gain access to the chemical materials. Can that be right? Is it as simple as mixing a solution of GFP or lucerfase in a dish with the fertilized eggs and a reagent (which one? lipid based maybe?)and letting them sit at room temp for a while? Or just using a micropipette to directly inject the lucerfase or gfp genes into the egg before it starts dividing? Can this be done by hand? I've got to be missing something here, it can't be as easy as it seems from my admittedly cursory research... is it? Would the effect be likely to be expressed throughout the body, especially the skin? Or would it be a mixed bag of animals showing different levels of expression? Would the effect be temporary or permanent? I assume it would be germ-line and the animals would be capable of breeding normally and passing on the introduced genes at various levels of expression to their offspring right?

 

Thanks in advance for any help exploring this. If anyone wants to post a detailed "how to", or PM me one on the easiest way to do this feel free

 

Sincerely,

Dave

Do you believe that you have the right to create an organism that can never hide from predators, that thus can never have an evolutionary niche 

[Moontanman]

27 minutes ago, Olin said:

Do you believe that you have the right to create an organism that can never hide from predators, that thus can never have an evolutionary niche 

We are talking about captive animals, captive animals are not part of nature nor should they be released back into nature. I see no ethical problems with this.... 

Edited November 13, 2018 by Moontanman

[CharonY]

In the last 8 years or so, services have popped up where you can design and have them made the required constructs (while not terribly cheap, it makes since easier).

However, you still need to do a successful viral transfection and that means setting everything up for sterile cell culture.  Unless I missed some major development, that is still rather labour intensive and prone to contamination issues, especially in home settings without training. Can one be theoretically successful? Sure, but it will still take time and money. It is one of the reasons why despite a lot of internet hype in the DIY area there are not regular posts of folks with their glowing frogs. Getting GFP producing coli is much easier, but even then there will be challenges (mostly contamination and risk of growing certain pathogens).

[Endy0816]

This little guy was actually found to naturally fluoresce since this thread started.

https://en.wikipedia.org/wiki/Polka-dot_tree_frog

And would not naturally show up like this. I think even most of our GM critters wouldn't.  Black light requirement, be easy enough to provide for a pet enclosure though.

Edited November 13, 2018 by Endy0816

[CharonY]

3 hours ago, Endy0816 said:

This little guy was actually found to naturally fluoresce since this thread started.

There are plenty of naturally fluorescent animals. GFP is only one of the man fluorophores around. Another well-known and common examples are scorpions, which you can also get in certain pet shops. 

[Endy0816]

4 hours ago, CharonY said:

There are plenty of naturally fluorescent animals. GFP is only one of the man fluorophores around. Another well-known and common examples are scorpions, which you can also get in certain pet shops.

Yeah this is just the frog they found that does so. We can still debate the broader issues. As for the frog itself, we're good.

[Olin]

18 hours ago, Moontanman said:

We are talking about captive animals, captive animals are not part of nature nor should they be released back into nature. I see no ethical problems with this.... 

So you believe that you have the right to to create whatever sick form of life that you choose for your pleasure.....

Why do you imprison things

17 hours ago, Endy0816 said:

This little guy was actually found to naturally fluoresce since this thread started.

https://en.wikipedia.org/wiki/Polka-dot_tree_frog

And would not naturally show up like this. I think even most of our GM critters wouldn't.  Black light requirement, be easy enough to provide for a pet enclosure though.

Is looking at frogs in a tank fun

[Moontanman]

18 minutes ago, Olin said:

So you believe that you have the right to to create whatever sick form of life that you choose for your pleasure.....

I did not say that, your attempt to defame is pitiful... 

18 minutes ago, Olin said:

Why do you imprison things

I like breeding captive animals, some of which are endangered, I have the proper permits and my animals receive better care and live longer lives than their wild counterparts. 

18 minutes ago, Olin said:

Is looking at frogs in a tank fun

It can be educational and satisfying to see animals up close that you would never get to see in the wild and yes I enjoy it but I don't keep frogs... 

[CharonY]

8 hours ago, Endy0816 said:

Yeah this is just the frog they found that does so. We can still debate the broader issues. As for the frog itself, we're good.

Oh right the question in OP,  forgot about that.

[Olin]

26 minutes ago, Moontanman said:

I did not say that, your attempt to defame is pitiful... 

I like breeding captive animals, some of which are endangered, I have the proper permits and my animals receive better care and live longer lives than their wild counterparts. 

It can be educational and satisfying to see animals up close that you would never get to see in the wild and yes I enjoy it but I don't keep frogs... 

Bio luminescent frogs are not endangered. They are sad toys for sadder people

[Moontanman]

2 minutes ago, Olin said:

Bio luminescent frogs are not endangered. They are sad toys for sadder people

I would disagree with you on that, frogs worldwide are endangered to some extent. Do you have pets? 

[Olin]

1 hour ago, Moontanman said:

I would disagree with you on that, frogs worldwide are endangered to some extent. Do you have pets? 

Bio luminescent frogs are not endangered unless you consider foolish peoples unethical experiments deserving of endangered species protection.

I have hunting dogs that are never leashed and I let run free in national forest as often as I can.  They also kill rabbits in my yard. They have never once been in an aquarium

[Endy0816]

1 hour ago, Olin said:

Bio luminescent frogs are not endangered unless you consider foolish peoples unethical experiments deserving of endangered species protection.

I have hunting dogs that are never leashed and I let run free in national forest as often as I can.  They also kill rabbits in my yard. They have never once been in an aquarium

You are still restricting them in some fashion though. Same concept as a tank(paludarium really). One with different bioluminescent and fluorescent species would be particularly fascinating.

...and the picture above is of a natural species of frog. No ethical issues on that front.

[Olin]

19 minutes ago, Endy0816 said:

You are still restricting them in some fashion though. Same concept as a tank(paludarium really). One with different bioluminescent and fluorescent species would be particularly fascinating.

...and the picture above is of a natural species of frog. No ethical issues on that front.

Just because one can, does not mean one should

[Endy0816]

1 hour ago, Olin said:

Just because one can, does not mean one should

There's valid reasons one might do so though. Aesthetics or to better appreciate nature.

[Moontanman]

6 hours ago, Olin said:

Bio luminescent frogs are not endangered unless you consider foolish peoples unethical experiments deserving of endangered species protection.

I have hunting dogs that are never leashed and I let run free in national forest as often as I can.  They also kill rabbits in my yard. They have never once been in an aquarium

Do they roam at will? Or do you keep them inside a fence? Why are your animals any better or worse than mine? You claim such experiments are unethical, how do you think hunting dogs were breed? Do you think a wolf suddenly gave birth to a hunting dog who decided to live with humans? Dogs were created by people through selective breeding as are all domestic animals, why are they any more ethical? 

[Olin]

28 minutes ago, Moontanman said:

Do they roam at will? Or do you keep them inside a fence? Why are your animals any better or worse than mine? You claim such experiments are unethical, how do you think hunting dogs were breed? Do you think a wolf suddenly gave birth to a hunting dog who decided to live with humans? Dogs were created by people through selective breeding as are all domestic animals, why are they any more ethical? 

Do you enjoy watching your frogs in an aquarium?  Some dogs actually fit what you said, my neighbor across the street has a pit bull, seems nice enough but it will never be unleashed not even once in it's life.  My dogs get to run free and chase deer as wolves once did, it's beautiful really

https://imgur.com/a/HvNT7Oy

[Moontanman]

1 hour ago, Olin said:

Do you enjoy watching your frogs in an aquarium?  Some dogs actually fit what you said, my neighbor across the street has a pit bull, seems nice enough but it will never be unleashed not even once in it's life.  My dogs get to run free and chase deer as wolves once did, it's beautiful really

https://imgur.com/a/HvNT7Oy

I've already said I don't have frogs... It's unethical to allow domestic animals to run wild animals for fun... 

[Phi for All]

!

Moderator Note

It's been questioned through the Report feature whether the ethics of genetic manipulation are a hijack of the thread. Staff feels it's fair ground and not off-topic.

That said, since this is a science site, some evidence would go a long way in supporting what has been up to now a very emotional argument from incredulity. It would be best to offer arguments against this genetic practice in particular, and not just a generalized animals-in-cages-is-wrong rant. I think that case has been expressed well enough, and now needs some support rather than further repetition.