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Author: Subject: DIY GMO fish and amphibians...
tantric2.718
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[*] posted on 7-1-2017 at 16:59
DIY GMO fish and amphibians...


I remember hearing in a lecture circa 95 the details of how DNA is organized in fertilized fish eggs and thinking - you can just inject the DNA? No vector no problem? Why don't I have glow in the dark goldfish? Well, it took another decade, but it did happen. In addition to glofish, there are now pink angelfish (angelfish are cichlids, a clade of relatively intelligent fish given to paternal care, much better than danios). I read that it takes 10,000 tries to get a correct insertion, in the angelfish case, but that's also doable....
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Sulaiman
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[*] posted on 7-1-2017 at 17:33


why ?

I understand the persuit of pure research,
but try to do something more useful than making pink fish,

P.S any spelling errors are due to my psychic cactus inyerface.


You seem mad enough ... welcome




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tantric2.718
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[*] posted on 7-1-2017 at 19:52


before there were glofish, this was the standard:




dye injected fish. you can still get 'tuttifrutti' dwarf frogs...


TuttiFruttiFrog_-_segrestfarms-com.jpg - 64kB



also dye injected. a scientist in taiwan created gmo pink angelfish, but never marketed them.




there are GFP axolotls you can buy....


9858357.jpg - 12kB

frankly, it's a wide open market, with lots of potential, and it's cool. in the 90s when i was one of those deplorable rave monkeys, i taught myself o-chem and set up a nonprofit mdma lab, just so my peeps wouldn't poison themselves with crap pills. got busted red handed and got off with an invalid search warrant AKA karma.
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[*] posted on 7-1-2017 at 20:08


This actually interests me a lot. To answer your question, direct microinjection of DNA into the pronucleus of a zygote can result in incorporation of the DNA into the genome. However, this process suffers from very low efficiency, and the incorporation site is random. A more modern technique is to use CRISPR/Cas9 to cause a double-strand break at a particular site, and introduce DNA flanked with sequences identical to those around the break. Homology-directed repair will then cause incorporation of the DNA into the site of the break.

On a related topic:
I am seriously investigating the feasibility of genetically engineering a human. I am particularly interested in having skin cells express a photolyase gene, to protect against UV damage and skin cancer. So far, I believe this is possible with current technology. However, this will undoubtedly require the collaboration of multiple researchers, and funding is an issue.




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[*] posted on 8-1-2017 at 02:56


You will also need approval of an ethical committee if you intent to try this with actual people. Experiments like that are not without risk. You will need to prove that the benefits to science/health outweigh the risks to your test subjects.
And to be honest, your chances for getting approval for this idea are not high. As far as I know, genetic modification of humans has only been been done so far for patients that suffered from fatal and otherwise untreatable diseases.

It would be doable to demonstrate your idea in cell culture though. No permission needed.




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[*] posted on 8-1-2017 at 09:37


a patent I found on transgenic sea monkeys...

https://www.sciencemadness.org/whisper/viewthread.php?tid=26...




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tantric2.718
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[*] posted on 10-1-2017 at 15:40


Quote: Originally posted by Metacelsus  
This actually interests me a lot. To answer your question, direct microinjection of DNA into the pronucleus of a zygote can result in incorporation of the DNA into the genome. However, this process suffers from very low efficiency, and the incorporation site is random. A more modern technique is to use CRISPR/Cas9 to cause a double-strand break at a particular site, and introduce DNA flanked with sequences identical to those around the break. Homology-directed repair will then cause incorporation of the DNA into the site of the break.

On a related topic:
I am seriously investigating the feasibility of genetically engineering a human. I am particularly interested in having skin cells express a photolyase gene, to protect against UV damage and skin cancer. So far, I believe this is possible with current technology. However, this will undoubtedly require the collaboration of multiple researchers, and funding is an issue.


i read about microinjection - 1 in 10,000. my point was that microinjection could be done in a garage lab. with patience. how does CRISPR work? could you still use microinjection but with this, put the genes in the right place?

i just read the seamonkey patent wtf is a 'gene blast gun'? and where can i get one?

[Edited on 10-1-2017 by tantric2.718]
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Metacelsus
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[*] posted on 10-1-2017 at 18:25


"Gene gun" refers to biolistics, where dense particles coated with DNA are literally shot at a tissue sample.
https://en.wikipedia.org/wiki/Gene_gun

For a short guide to CRISPR/Cas9, see: https://www.addgene.org/crispr/guide/




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[*] posted on 10-1-2017 at 22:08


If someone in really interested in this kind of stuff, begin at the beginning, with something made competent (able to take up DNA) easily. Bacteria for example. Start with E. coli for plasmid transformations and B. subtilis for genomic integration. I think these two are doable for an amateur, but also tricky enough to be very valuable in the learning process.
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[*] posted on 11-1-2017 at 05:46


I've done work transforming E. coli in an academic lab, and it would definitely be possible in an amateur setting (although most of the starting materials would have to be purchased first).

[Edited on 1-11-2017 by Metacelsus]




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[*] posted on 11-1-2017 at 06:08


Electroporation looks promising:

http://www.celetrix.com/alone/alone.php?id=45

O3




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[*] posted on 11-1-2017 at 09:00


Electroporation is nice to transform cells that are not chemically or naturally competent, for example Staphylococcus aureus, or when high efficiency is needed (this requires a highly optimised protocol though).

For E. coli and B. subtilis chemical (Ca2+ transformation) and natural (aminoacid starvation) competence respectivally are fine for standard transformations. The standard methods are easier as you don't need electroporation wells and a high voltage power source.

[Edited on 11-1-2017 by Tsjerk]
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[*] posted on 11-1-2017 at 09:38


It is also called transfection.

I have worked for a little time for Lonza Bioscience and I was a seller amongst other things for a transfection device nucleofector.

It allows incorporation of DNA into eucariotic cells or other cells without the use of viral vectors and thus allows one to bypass the need for special biochemical level labs accrediation (using viruses).
From what I remember you may finetune the voltage pulse shape, the intensity aswel as the transfection medium properties to optimise the % of successful transfection and addapt to specific type of cells...a big deal of the cells doesn't survive the shock and some like neurons are very sensitive!

There are two types of devices complementary: one for adherent cells (like neurons) and one for floating cells...difference resides into the shape of the electrodes.

[Edited on 11-1-2017 by PHILOU Zrealone]




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[*] posted on 11-1-2017 at 10:12


You are right about the transfection, people working with pro-caryotes call it transformation and "eu's" call it transfection although they are the same.
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[*] posted on 12-1-2017 at 04:00


Quote: Originally posted by Sulaiman  
why ?

I understand the persuit of pure research,
but try to do something more useful than making pink fish,


Why not? You know the old saying - Pink fish today, intelligent, fast, strong, super humans tomorrow!


I think it's amazing. I would like to see humans have the gene from sharks that lets them regrow teeth after they have fallen out.... although we would have to make sure there were no aggressive bitey side effects. ;-)




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tantric2.718
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[*] posted on 16-1-2017 at 04:58


how about yeast? custom yeast for microbrewers would sell like hot cakes.
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[*] posted on 16-1-2017 at 07:10


I've transformed and made GMO yeasts a lot. Mostly Saccharomyces cerevisiae (bakers yeast), but also Pichia pastoris and other more obscure yeasts.
S. cerevisiae is not very difficult at all to work with. Certainly doable in an amateur setting.

However, I would expect making or buying the DNA constructs to do what you want is going to be more of a challenge.
Never mind figuring out what modifications to make to achieve whatever goal you have in mind.
Some yeasts have so many copies of genes and/or use variations of the genetic code that it makes them time-consuming/annoying to work with. Still possible, just a lot more work.

I'm not convinced your strains would 'sell like hot cakes', nor would it be trivial to modify yeasts in a particularly useful way (for brewers purposes).




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[*] posted on 16-1-2017 at 09:37


found this...

Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

working on zebrafish (basal species for glofish) using microinjection....but with CRISPR/cas9 technology. now - wow, this is promising...
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[*] posted on 16-1-2017 at 11:11


You might be interested in this guide from AddGene:

Attachment: CRISPR_101_eBook_small.pdf (4.1MB)
This file has been downloaded 488 times





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[*] posted on 1-3-2017 at 11:46


Any of you guys have a procedure for transfection for e coli, and a general list of materials? We literally just started covering genetic engineering in my biology class, and this is fascinating.
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[*] posted on 1-3-2017 at 13:22


Quote: Originally posted by Ursa  
Any of you guys have a procedure for transfection for e coli, and a general list of materials? We literally just started covering genetic engineering in my biology class, and this is fascinating.


If your school has access, Current Protocols in Molecular Biology is a one-stop shop for this sort of information:
http://onlinelibrary.wiley.com/book/10.1002/0471142727/homep...

I've attached the sections I keep on hand when I'm transforming bacteria; if there's interest in more, ask in References and I'll see what I can do.


Attachment: Digestion of DNA with Restriction Endonucleases.pdf (203kB)
This file has been downloaded 79 times

Attachment: Resolution and recovery of large DNA fragments - agarose gel electrophoresis.pdf (142kB)
This file has been downloaded 56 times

Attachment: Enzymatic Amplification of DNA by PCR- Standard Procedures and Optimization.pdf (175kB)
This file has been downloaded 55 times

Attachment: Minipreps of Plasmid DNA.pdf (255kB)
This file has been downloaded 71 times

Attachment: Introduction of Plasmid DNA into Cells.pdf (146kB)
This file has been downloaded 62 times




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[*] posted on 2-3-2017 at 09:00


Quote: Originally posted by mayko  
Quote: Originally posted by Ursa  
Any of you guys have a procedure for transfection for e coli, and a general list of materials? We literally just started covering genetic engineering in my biology class, and this is fascinating.


If your school has access, Current Protocols in Molecular Biology is a one-stop shop for this sort of information:
http://onlinelibrary.wiley.com/book/10.1002/0471142727/homep...

I've attached the sections I keep on hand when I'm transforming bacteria; if there's interest in more, ask in References and I'll see what I can do.











Thanks man, helps a lot.




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[*] posted on 7-3-2017 at 08:38


Oh yeah, general question, say I wanted a luminescence gene added to fish, would I be able to simply separate that from a known bioluminescent bacteria, and put that into a plasmid, such as Photobacterium phosphoreum, or would I have to purchase a plasmid with that gene already insterted? How would I go about doing either of those? I know how to do the cultivation, but how would I insert it into a plasmid? CRISPR at a specific set of genes/ polymerase and ligase general reaction?, I get how to introduce the plasmid into a bacterium, and how to introduce that into a fish egg culture. Also would I use fertilized eggs, or unfertilized eggs? I'm pretty sure its fertilized eggs, but just checking.



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[*] posted on 7-3-2017 at 16:40


Briefly, a common way to prepare the plasmid would be as follows. I am skipping a lot of details and small steps so as to provide an overview of the procedure. I am sure you will be able to find detailed tutorials/explanations online for each of the steps.

1. Isolate genomic DNA from the luminescent bacterium (culture bacteria, isolate DNA)
2. use PCR to make lots of copies of the luminescence gene
3. Ligate the PCR product into a plasmid that you can propagate in E coli
4. Transform E coli with the ligation mixture
5. Isolate E coli colonies that contain the right gene by restriction tests+electrophoresis (verify the size of the insert) and preferably sequencing the insert (rule out mutations in sequence introduced by PCR).
6. Culture more of the E coli containing the correct plasmid (lets call it pUrsa1), and isolate enough plasmid DNA from it for the next steps
7. Cut the Luminescence gene from pUrsa1, and at the same time cut open a suitable target plasmid (suitable for expressing the protein in your organism of interest) with the same set of restriction enzymes. The target plasmid is usually bought or obtained from a friendly colleague.
8. Isolate the opened target plasmid and the insert containing the luminescence gene by electrophoresis, cutting the bands from the gel and isolating the DNA from the pieces of gel.
9. Ligate the insert into the target plasmid
10. Transform E coli with it
11. Select colonies containing the correct plasmid (verify by cutting it with restriction enzymes and electrophoresis). Unlike PCR, restriction and ligation reactions only very rarely introduce mutations, so no need to sequence it again.
12. Again, scale up the culture of the E coli containing the right plasmid and isolate the plasmid DNA from it.

However, you can order a plasmid containing an insert of any random sequence from companies that makes synthetic plasmids. For the cost of those (expect around Eur 300,-) you can't even order the primers, enzymes, etc, you need. Never mind your time. Also, the convenience of being able to design your insert with any random sequence (restriction sites, immune tags, etc) and having it arrive in two weeks while you are free to do other useful things can't be beat so I highly recommend going that route.

I have no experience transforming fish. Your posts suggests to me that you should study how to transfer and express the gene in your target organism (ie the fish) a bit more.

[Edited on 8-3-2017 by phlogiston]




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[*] posted on 8-3-2017 at 11:16


Quote: Originally posted by phlogiston  
Briefly, a common way to prepare the plasmid would be as follows. I am skipping a lot of details and small steps so as to provide an overview of the procedure. I am sure you will be able to find detailed tutorials/explanations online for each of the steps.

1. Isolate genomic DNA from the luminescent bacterium (culture bacteria, isolate DNA)
2. use PCR to make lots of copies of the luminescence gene
3. Ligate the PCR product into a plasmid that you can propagate in E coli
4. Transform E coli with the ligation mixture
5. Isolate E coli colonies that contain the right gene by restriction tests+electrophoresis (verify the size of the insert) and preferably sequencing the insert (rule out mutations in sequence introduced by PCR).
6. Culture more of the E coli containing the correct plasmid (lets call it pUrsa1), and isolate enough plasmid DNA from it for the next steps
7. Cut the Luminescence gene from pUrsa1, and at the same time cut open a suitable target plasmid (suitable for expressing the protein in your organism of interest) with the same set of restriction enzymes. The target plasmid is usually bought or obtained from a friendly colleague.
8. Isolate the opened target plasmid and the insert containing the luminescence gene by electrophoresis, cutting the bands from the gel and isolating the DNA from the pieces of gel.
9. Ligate the insert into the target plasmid
10. Transform E coli with it
11. Select colonies containing the correct plasmid (verify by cutting it with restriction enzymes and electrophoresis). Unlike PCR, restriction and ligation reactions only very rarely introduce mutations, so no need to sequence it again.
12. Again, scale up the culture of the E coli containing the right plasmid and isolate the plasmid DNA from it.

However, you can order a plasmid containing an insert of any random sequence from companies that makes synthetic plasmids. For the cost of those (expect around Eur 300,-) you can't even order the primers, enzymes, etc, you need. Never mind your time. Also, the convenience of being able to design your insert with any random sequence (restriction sites, immune tags, etc) and having it arrive in two weeks while you are free to do other useful things can't be beat so I highly recommend going that route.

I have no experience transforming fish. Your posts suggests to me that you should study how to transfer and express the gene in your target organism (ie the fish) a bit more.

[Edited on 8-3-2017 by phlogiston]


Thank you very much. This website has been helping a lot already lol.




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