Gene Editing

Don Lee, Presenter Author

02/22/2016 Added

1407 Plays

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This video provides information on genetic transformation. For more information visit the Enviropig website: https://ge.unl.edu/enviropig/

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[00:00:03.773]Hello, this is Don Lee,
[00:00:05.711]and in Step two of the Enviropig story
[00:00:09.553]we're learning about how scientists like
[00:00:11.348]Brett White, who's pictured here,
[00:00:14.240]used technology to transform pigs,
[00:00:18.663]to introduce genetic changes
[00:00:21.648]in order to give the pig a characteristic
[00:00:25.003]that it didn't have before,
[00:00:27.522]but this technology is
[00:00:31.078]a changing world that scientists like Brett White
[00:00:34.827]are continually trying to work on and improve,
[00:00:38.519]so before we meet Brett White,
[00:00:41.758]and see how he works in the lab
[00:00:44.022]I wanted to give you an opportunity to think
[00:00:46.518]a little bit more about what he does,
[00:00:50.186]and how that impacts activities at
[00:00:52.112]the cell and molecular level in pigs.
[00:00:56.048]We're gonna compare these three kinds of
[00:00:58.368]genetic modifications here in the next few minutes.
[00:01:00.969]Mutations, genetic modification by
[00:01:03.650]the addition of a transgene,
[00:01:06.111]and then this new technology that
[00:01:08.942]Dr. White will refer to that's called gene editing.
[00:01:12.994]I think if you do this,
[00:01:15.397]you'll do a better job of imagining
[00:01:17.371]what's going on inside
[00:01:20.152]the cells of the pigs
[00:01:22.115]that Dr. White works with.
[00:01:25.714]Let's start with mutations.
[00:01:27.594]We're gonna get you to think about this molecule.
[00:01:30.404]The double-stranded DNA molecule
[00:01:32.948]that is the molecular
[00:01:36.396]bio-molecule that provides
[00:01:39.554]information for controlling traits.
[00:01:43.874]Genes are parts of chromosomes,
[00:01:45.754]and one gene would have the specific
[00:01:48.773]information to tell your cells, or a pig cells,
[00:01:52.035]how to assemble individual amino acids
[00:01:55.585]into a specific order and make a protein
[00:01:58.801]that has a structure that gives it
[00:02:01.518]the ability to control a specific trait,
[00:02:04.443]so genes encode proteins.
[00:02:06.323]Proteins control traits is the key idea,
[00:02:09.424]and so those traits could
[00:02:13.364]involve any of the specific details
[00:02:17.079]that have to happen inside
[00:02:18.704]the cells of the pig as it grows,
[00:02:20.886]and develops and becomes an adult pig,
[00:02:23.719]and completes its life cycle.
[00:02:25.983]Now what can happen is changes can occur
[00:02:28.711]in the DNA coding information,
[00:02:31.822]and those changes can alter the proteins that
[00:02:36.187]that gene is designed to encode,
[00:02:38.149]and that alteration can, therefore,
[00:02:40.586]result in an altered trait.
[00:02:44.302]The traits can be traits
[00:02:48.634]that might involve
[00:02:51.496]small subtle changes
[00:02:53.899]in like coat pigmentation,
[00:02:56.302]or maybe they would be economically
[00:02:58.879]important traits like growth rate.
[00:03:01.375]It all depends on the protein.
[00:03:03.279]It depends on the nature of
[00:03:04.695]the change in the protein.
[00:03:06.181]It depends on where that mutation occurred
[00:03:09.164]in this random process.
[00:03:12.241]Without mutations you don't have
[00:03:14.643]genetic variability amongst pigs,
[00:03:17.081]and you don't have the opportunity
[00:03:19.794]to make new combinations of pigs
[00:03:23.975]that provide a better animal
[00:03:27.102]for people to raise.
[00:03:29.749]Mutations are just a part of life.
[00:03:33.048]Now the thing to keep in mind is that
[00:03:35.207]pigs like a lot of other living things
[00:03:37.425]have tens of thousands of genes,
[00:03:39.468]and these are organized along these long
[00:03:42.869]DNA molecules that make up the chromosome,
[00:03:45.829]and it turns out that mutations are random.
[00:03:49.590]We don't know which gene, a mutation,
[00:03:51.541]a mistake in DNA replication,
[00:03:53.769]or a chemically induced mutation will occur in,
[00:03:57.822]what genes are affected by a mutation,
[00:04:00.247]and how a specific mutation
[00:04:02.802]will change the gene,
[00:04:05.044]so the random nature of mutations
[00:04:07.946]results in far less control
[00:04:12.074]for the geneticists and the breeders.
[00:04:15.173]They have to sort through mutations,
[00:04:17.486]and determine which ones might have
[00:04:20.087]a positive benefit
[00:04:23.855]for the purpose of raising pigs as a meat animal.
[00:04:29.091]So mutations is one way in which
[00:04:31.134]genetic change can occur.
[00:04:33.521]Another way in which genetic change can occur,
[00:04:36.242]and the method that we're focusing on
[00:04:38.309]learning about is transgenes.
[00:04:40.549]Here is a diagram that summarizes
[00:04:43.209]the steps we're learning about in
[00:04:46.392]pig transformation or pig genetic engineering.
[00:04:50.037]The first steps involve isolating
[00:04:52.916]the DNA molecules from the organisms
[00:04:56.038]that contain genes that will either
[00:04:58.999]control gene expression,
[00:05:00.415]or encode the protein,
[00:05:02.493]and, therefore, modify the trait,
[00:05:05.071]or introduce the trait that
[00:05:06.615]we're interested in,
[00:05:07.659]so in the case of enviropigs
[00:05:09.610]the gene from the microorganism,
[00:05:12.675]from the bacteria encoded
[00:05:14.613]a protein called phytase,
[00:05:16.854]and then a part of the gene from the mouse
[00:05:19.396]encoded an on/off switch, a promoter
[00:05:22.751]that turned that gene on in the saliva cells,
[00:05:26.157]so that the protein was made at
[00:05:28.177]the right stage of the pigs digestion process,
[00:05:31.733]so what we're learning about now is Step two,
[00:05:34.414]how this modified gene is actually introduced
[00:05:37.793]into the embryonic cells of the pigs.
[00:05:42.088]We're gonna focus on what happens
[00:05:44.108]at this stage of the genetic engineering process.
[00:05:48.090]This is the work done by scientists like Brett White.
[00:05:51.410]Now the key is they have to have
[00:05:53.813]a delivery system.
[00:05:55.346]They use a very fine needle that's introduced
[00:05:58.712]into this embryonic cell.
[00:06:00.767]It could be a fertilized egg,
[00:06:02.288]the very start of creating
[00:06:04.505]a new generation of pigs,
[00:06:06.873]and they have to target then the nucleus
[00:06:09.868]because that's where the chromosomes,
[00:06:11.528]and the DNA molecules that
[00:06:13.003]we're envisioning are located.
[00:06:15.464]That's where they have to
[00:06:18.249]focus their attention,
[00:06:20.756]and so what goes on then is
[00:06:22.996]they will introduce copies of
[00:06:25.945]the designed transgenes.
[00:06:28.034]We learned that they combine that promoter
[00:06:29.951]with the coding region,
[00:06:31.587]so that the transgene would have
[00:06:33.653]the effect desired in
[00:06:37.067]the genetically engineered pigs.
[00:06:39.249]What they'll do is incorporate lots of copies,
[00:06:42.571]or introduce lots of copies of
[00:06:44.847]this designed transgene into
[00:06:47.366]the nucleus of those embryonic cells,
[00:06:49.769]and then hope that some sort of
[00:06:53.241]recombination event takes place where
[00:06:55.899]the existing pig chromosome breaks,
[00:06:58.413]and that allows for the new transgene DNA
[00:07:02.476]to get incorporated into
[00:07:04.820]the chromosome during a repair process,
[00:07:07.619]and it's not entirely understood how that works,
[00:07:11.600]but if you introduce enough copies
[00:07:14.457]somehow you can increase the odds that
[00:07:17.939]that transgene will get inserted,
[00:07:20.765]and once it's there it can work its design.
[00:07:23.760]It will be turned on in the right cell
[00:07:25.339]at the right time.
[00:07:26.545]It will encode the right amino acid
[00:07:28.099]sequence of the protein,
[00:07:29.817]and it will introduce the ability
[00:07:31.791]to make this protein into your
[00:07:34.472]genetically engineered pigs,
[00:07:36.457]and they will become enviropigs.
[00:07:39.453]In this case the ability to better digest
[00:07:43.377]the phosphorus-containing molecules in the food
[00:07:47.010]that farmers provide for them,
[00:07:50.936]but, again, what we have to keep in mind
[00:07:53.594]is genes are a part of chromosomes,
[00:07:57.332]and we're trying to introduce this transgene
[00:07:59.596]somewhere into an existing pig chromosome.
[00:08:03.253]It turns out that the insertion process
[00:08:07.108]that I just described is random.
[00:08:10.311]The genetic engineer using the method
[00:08:13.423]that was used to create enviropigs
[00:08:16.011]were reliant on finding pigs
[00:08:19.283]that got a gene insertion,
[00:08:21.338]but not knowing exactly where that
[00:08:23.277]gene insertion went,
[00:08:25.877]so while they could design how the gene worked
[00:08:28.919]they had less control over where
[00:08:32.559]the gene inserts, and how it inserts.
[00:08:36.924]In other words, did one copy of the gene insert,
[00:08:39.688]or did multiple copies of the gene insert.
[00:08:42.219]Did it insert in such a way that
[00:08:44.087]it would be functional in the pig cells,
[00:08:48.302]so the method of
[00:08:51.839]gene introduction
[00:08:54.625]that was used in enviropig was the standard,
[00:08:58.442]was the way genetic engineering was done
[00:09:01.808]at that time in animals,
[00:09:03.688]and it still is today,
[00:09:05.367]but that is all starting to change with
[00:09:08.018]some new technology called gene editing.
[00:09:10.603]Let's learn about that.
[00:09:12.437]The key with gene editing is that
[00:09:15.363]a gene has been discovered that
[00:09:17.626]originally occurs in a microorganism.
[00:09:20.227]This gene encodes what I'll call an editing protein.
[00:09:24.104]This editing protein
[00:09:26.692]could have a specific name.
[00:09:28.631]I think Dr. White uses the name "CRISPR"
[00:09:32.066]to describe one type of
[00:09:34.944]DNA editing protein,
[00:09:37.963]but what all of these editing protein systems
[00:09:40.689]have in common are these capabilities.
[00:09:43.277]These genes occur in microorganisms naturally,
[00:09:46.818]and they use them in order to
[00:09:49.836]specifically target invading DNA
[00:09:53.575]that comes from viruses,
[00:09:55.374]so they have a part of the protein they encode.
[00:09:58.567]I'll use pictures here to represent
[00:10:00.912]the amino acid sequence
[00:10:04.029]that would make up this part of the protein.
[00:10:06.675]They have a part of the protein design
[00:10:08.695]to look for and detect
[00:10:11.714]a specific DNA sequence.
[00:10:13.676]I'll use this magnifying glass for that.
[00:10:16.857]They also have a part of this protein
[00:10:19.781]that allows it to act like a molecular scissors,
[00:10:23.578]and specifically cut the DNA,
[00:10:26.242]and then they have a part of the protein
[00:10:28.842]that can be involved in editing the DNA
[00:10:32.278]either removing nucleotides,
[00:10:35.122]or changing nucleotides, potentially,
[00:10:37.966]even introducing some additional
[00:10:40.799]nucleotides of DNA
[00:10:42.680]that don't already exist.
[00:10:44.723]So this gene encodes this complete protein
[00:10:49.011]that has these multiple functions,
[00:10:51.322]and then if that protein encounters
[00:10:53.864]a DNA molecule inside of the pig,
[00:10:57.509]or any other living thing
[00:10:59.588]the sequence can be examined
[00:11:03.166]by these editing proteins until
[00:11:06.753]the exact sequence targeted is found,
[00:11:09.528]and then the protein can make
[00:11:13.405]a specific edit by clipping the DNA,
[00:11:16.749]and either adding new DNA sequence,
[00:11:19.430]or changing the DNA sequence that's there,
[00:11:22.925]so now you have a very specifically edited gene.
[00:11:27.324]It's not nearly as random as
[00:11:30.740]the mutation method, or the transgene method.
[00:11:33.666]The edited gene can be targeted.
[00:11:35.906]You know exactly which gene
[00:11:37.346]is gonna be changed,
[00:11:38.867]and then you can as the genetic engineer
[00:11:41.061]design this CRISPR protein,
[00:11:43.249]or this DNA editing protein to give the gene
[00:11:46.754]a new function, or enhance its function,
[00:11:49.367]or even to potentially make a change
[00:11:52.756]that knocks out this gene,
[00:11:55.357]so it has no function.
[00:11:57.470]So the big difference here
[00:12:00.010]is that if you want to make
[00:12:01.986]a genetically edited version of a pig
[00:12:05.687]instead of introducing a new DNA
[00:12:08.550]you can introduce the specifically
[00:12:10.628]designed CRISPR proteins.
[00:12:12.683]They will target the nucleus.
[00:12:15.159]You can inject them into the nucleus,
[00:12:17.027]and once they're there
[00:12:18.385]they can go and do the work
[00:12:20.104]they're designed to do.
[00:12:21.323]Target a specific gene and edit it.
[00:12:25.381]The steps involving gene editing
[00:12:29.165]are gonna be a little bit different than
[00:12:31.162]the steps involving making a transgenic pig,
[00:12:34.447]so let me make the changes.
[00:12:36.363]You still obtain a new DNA sequence,
[00:12:39.787]a new gene from a microorganism,
[00:12:42.787]but instead it would be a gene that encodes
[00:12:45.015]these DNA editing proteins,
[00:12:47.070]and you could design the DNA editing
[00:12:49.810]to specifically target an enzyme,
[00:12:53.409]a gene encoding an enzyme,
[00:12:55.011]and you could then try to turn
[00:12:56.546]that into a phytase enzyme.
[00:13:00.174]You may have to add some
[00:13:01.792]additional DNA sequences
[00:13:03.394]to help you with that editing process,
[00:13:05.832]but the key then is you add the protein,
[00:13:08.194]maybe some additional DNA during
[00:13:10.655]the transformation step
[00:13:12.675]into these embryonic pig cells.
[00:13:16.170]Once they're modified
[00:13:18.863]they're placed in a surrogate mother
[00:13:22.241]where potentially
[00:13:24.914]gene edited pigs are born,
[00:13:27.771]and then those gene edited pigs can be bred
[00:13:31.172]to get your desired result,
[00:13:33.645]so gene editing has the potential to change
[00:13:36.872]the genetic engineering process
[00:13:38.869]because it's more precise,
[00:13:40.866]and more specific,
[00:13:43.188]and doesn't introduce an entire new gene
[00:13:46.009]into a living organism.

Gene Editing

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Gene Editing

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