Dendro Dave

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?

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