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RE: research
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by GREGLONGHURST on April 25, 2006
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DNA can be obtained from a shed skin. This is probably the easiest & least stressful to the snake. Personally, I don't see how any kind of a determination about venom can be made from this, but that's what was asked for.
~~Greg~~
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RE: research
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by BGF on April 25, 2006
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This would be a poor choice of material for the stated project as it would only reveal genome sequences, but not inform as to introns vs exon regions without constructing a venom gland cDNA library. Full genome sequence is not needed, only the mRNA transcripts. Even this would necessitate the production of synthetic genes.
Here are some literature references I would suggest having a read of.
Pergolizzi RG, Dragos R, Ropper AE, Menez A, Crystal RG. Related
Protective immunity against alpha-cobratoxin following a single administration of a genetic vaccine encoding a non-toxic cobratoxin variant.
Hum Gene Ther. 2005 Mar;16(3):292-8.
Harrison RA
Development of venom toxin-specific antibodies by DNA immunisation: rationale and strategies to improve therapy of viper envenoming.
Vaccine. 2004 Apr 16;22(13-14):1648-55
Cheers
Bryan
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RE: research
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by pspinkey on April 25, 2006
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You hit the nail on the head with wanting venom gland tissue samples. It unfortuanately will cause more pain for the animals which is something I really do not want to do. The other idea is to "shotgun" clone as its known in my area lol. This would require just skin but would create a lot more work for me and increase the length of my research. As far as validity of my ideas research seems to point that is possible but hasnt fully been done. I am hoping to use different promoters to get the proper mix of proteins. If Im mistaken on anything let me know
Chad Kirk
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RE: research
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by Cro on April 25, 2006
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Chad: Getting venom gland tissue samples could be quite possible, especially if you are willing to work with freshly road-killed animals.
I often come across road-killed rattlesnakes that have just been hit, and are still writhing on the road. It would be fairly easy to collect their heads and ice them down, and save them for a research project like this. I believe others would be willing to do this for you also.
Once you get your research set up, you should post again. It is very possible that you could obtain all the specimins you need from local collectors in your area, especially if you are willing to pay a small amount for them to collect road-killed rattlesnake heads for you, or to purchase the heads of snakes that died in captivity and were frozen.
It is far more likely you will be able to obtain help this way, than if you want tissue samples from living snakes in peoples collections.
Keep us informed on how you progress with this.
Best Regards JohnZ
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RE: research
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by BGF on April 26, 2006
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Snakes suffering from Dunlops Disease (aka: roadkills) won't work as the mRNA breaks down very quickly. Plus stimulating the snake by milking it a few days out greatly increases the mRNA levels. Using a venom gland is the only logical approach as that way you get actual toxin transcripts. Using genomic DNA would be a bad avenue as you'd have to figure out what was coding for a related normal body protein and which for a venom protein. Then you'd have to figure out exon/intron boundaries and go from there. Much simpler just to start with venom gland mRNA and use southern blotting to highlight the toxin colonies as opposed to ones consisting of intracellular proteins like calglandulin. Then you have to figure out how to get yeast to express it (or bacteria). Along with all the associated problems of codon bias and the fact that expressing a single protein is hard enough without trying to do a complex mixture all at once.
Or you could just milk a snake and inject it into a horse. Quicker, easier, cheaper and much more representative of the molecular diversity and stoichiometry of the toxin proteins.
Cheers
Bryan
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RE: research
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by GREGLONGHURST on April 26, 2006
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Dr. Fry: I had always thought that Dunlop Disease was one incurred by middle-aged men, mostly beer drinkers, whose belly has dun lopped over their belt buckle.
I did not really think that DNA from a shed skin would be of much help. I am glad you cleared that up.
~~Greg~~
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