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--- chapter_17 [2024/08/24 18:58] – [Viral vectors] mike
+++ chapter_17 [2025/05/16 21:52] (current) – [Viral vectors] mike
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-<typo fs:x-large>Chapter 17. Newer tools for reverse genetics</typo>
+<-chapter_16|Chapter 16^table_of_contents|Table of Contents^chapter_18|Chapter 18->
-It is useful to briefly describe some newer technologies for reverse genetics that give researchers a bigger toolbox for genetic analysis. Most of these tools are developed for use in mice, but generally they can be adapted to different model genetic organisms with some technical tweaks.
+<typo fs:x-large>Chapter 17. Newer tools for %%reverse genetics%%</typo>
+It is useful to briefly describe some newer technologies for reverse genetics that give researchers a bigger and more advanced toolbox for genetic analysis. Most of these tools are developed for use in mice, but generally they can be adapted to different model genetic organisms with some technical tweaks.
 ===== Gene knock-ins =====
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 Gene knock-ins are a variation of the classic gene knockout we saw in [[chapter_16|Chap. 16]]. In this method, instead of replacing an endogenous gene with something like $neo^R$, we replace it with a modified version of the gene. For instance, instead of going through the complex transgene/knockout combination strategy described in Chapters [[chapter_15|15]]-[[chapter_16|16]], you could in principle simply replace the mouse globin genes with knock-ins of the human globin genes. (When the studies described in Chapters [[chapter_15|15]]-[[chapter_16|16]] were originally done, the knock-in technique had not yet been invented.) This greatly reduces the complexity of the experimental design of the globin gene project. Furthermore, the level of expression of transgenes is often difficult to control precisely because transgenes often use upstream regulatory sequences that are removed from their normal chromosomal context (the regulatory sequences used may be missing some important enhancer sequences, for instance). By comparison, a knock-in allele usually will be expressed at very similar levels as the wildtype allele, because all the normal enhancer sequences that regulate the endogenous locus are still there.
-To understand how gene knock-ins work, we must first discuss a site-specific recombination system such as the Cre-$lox$ system from bacteriophage P1 (Fig. {{ref>Fig1}}). $loxP$ is a 34 bp DNA sequence from the genome of bacteriophage P1. Normally, 34 bp is not long enough to drive homologous recombination in mice. However, a bacteriophage P1 enzyme called Cre (also called Cre recombinase) can cause DNA recombination (essentially crossing over) to occur between two different $loxP$ sites. This results in the sequences in between the $loxP$ sites to be deleted, leaving behind a single $loxP$ site as a recombination scar.
+To understand how gene knock-ins work, we must first discuss a site-specific recombination system such as the Cre-$lox$ system from bacteriophage P1 (Fig. {{ref>Fig1}}). $loxP$ is a 34 bp DNA sequence from the genome of bacteriophage P1. Normally, 34 bp is not long enough to drive homologous recombination in mice. However, a bacteriophage P1 enzyme called Cre (also called Cre recombinase) can cause DNA recombination (essentially crossing over) to occur between two different $loxP$ sites. This results in the sequences between the $loxP$ sites being deleted, leaving behind a single $loxP$ site as a recombination scar.
 <figure Fig1>
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 </figure>
-To generate a gene knock-in, you would usually follow the normal gene knockout experimental approach, with a few modifications. For example, let's say you want to modify a codon in exon 2 of a gene (Fig. 17.2). Here is how you might design your targeting construct:
+To generate a gene knock-in, you would usually follow the normal gene knockout experimental approach, with a few modifications. For example, let's say you want to modify a codon in exon 2 of a gene (Fig. {{ref>Fig2}}). Here is how you might design your targeting construct:
   * You would clone (probably using PCR) DNA sequences from exon 1, intron 1, and exon 2.
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   * $tk^{HSV}$ would go after exon 2.
-This targeting construct can be used to generate a "knockout", but since neoR is located in an intron, it doesn't really disrupt the gene at all. This targeting construct will integrate via homologous recombination using sequences from exon 1 and exon 2 in the targeting construct, thereby bringing in your engineered mutation to the correct chromosomal location in the ES cells. Once you have targeted ES cells, you can remove the neoR gene from the intron by expressing Cre in the ES cells before creating chimeric mice, leaving behind a loxP scar in the intron. This is usually done with a separate transgene that expresses Cre.
+This targeting construct can be used to generate a "knockout", but since $neo^R$ is located in an intron, it doesn't really disrupt the gene at all. This targeting construct will integrate via homologous recombination using sequences from exon 1 and exon 2 in the targeting construct, thereby bringing in your engineered mutation to the correct chromosomal location in the ES cells. Once you have targeted ES cells, you can remove the $neo^R$ gene from the intron by expressing Cre in the ES cells before creating chimeric mice, leaving behind a $loxP$ scar in the intron. This is usually done with a separate transgene that expresses Cre.
-<figure>
+<figure Fig2>
 {{ :knock_in.jpg?400 |}}
 <caption>
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 ===== Conditional knockouts =====
-Conditional knockouts are a variation of the gene knock-in method (Fig. {{ref>Fig3}}). A conditional knockout is a gene knockout that only occurs under certain conditions, such as in specific cell types or at specific developmental time points. To create a conditional knockout, you would use the gene knock-in method above to add two $loxP$ sites in two different introns of a gene you wanted to knock out. Since the $loxP$ sites are in introns, they typically will not affect the function of the gene; this allele usually will be indistinguishable from wildtype. If you can somehow express Cre in these cells, then Cre will cause recombination to occur between the two loxP sites, which will delete all the exons in between. This usually results in a frameshift mutation, and frameshifts usually result in a null allele. In conditional knockout experiments, Cre is usually expressed as a transgene, and there are other tricks that can be done to express Cre only in specific cells (such as using [[chapter_17#Viral_vectors|viral vectors]], or to only express Cre under the control of an inducible promoter that depends on an external unducer, or some combination thereof.
+Conditional knockouts are a variation of the gene knock-in method (Fig. {{ref>Fig3}}). A conditional knockout is a gene knockout that only occurs under certain conditions, such as in specific cell types or at specific developmental time points. To create a conditional knockout, you would use the gene knock-in method above to add two $loxP$ sites in two different introns of a gene you wanted to knock out. Since the $loxP$ sites are in introns, they typically will not affect the function of the gene; this allele usually will be indistinguishable from wildtype. If you can somehow express Cre in these cells, then Cre will cause recombination to occur between the two $loxP$ sites, which will delete all the exons in between. This usually results in a frameshift mutation, and frameshifts usually result in a null allele. In conditional knockout experiments, Cre is usually expressed as a transgene, and there are other tricks that can be done to express Cre only in specific cells, such as using [[chapter_17#Viral_vectors|viral vectors]], or to only express Cre under the control of an inducible promoter that depends on an external inducer, or some combination thereof.
 <figure Fig3>
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 </figure>
-Conditional knockouts are useful for studying genes that are expressed in multiple tissues, but you only wish to study the effect in one kind of tissue. For instance, let's say you are studying gene X, which is expressed in both brain and liver tissue. When you knock out gene X using the standard approach, you find that the mice die as embryos due to a brain defect during embryogenesis. However, you are interested in the role of gene X in the livers of adult mice. You can generate a conditional knockout of gene X where it is only knocked out in adult livers but remains functional in embryonic brains so that they can be born and develop into adults that you can then study for liver function. One way to achieve this is to find a gene that is only expressed in adult livers, clone the upstream regulatory sequences that regulate expression in adult liver cells, and generate a transgene that expresses Cre under the control of this regulatory sequence. You can then generate a floxed allele (an allele with flanking $loxP$ sites) of gene X and breed mice that carry this allele together with your liver specific Cre transgene.
+Conditional knockouts are useful for studying genes that are expressed in multiple tissues, but you only wish to study the effect in one kind of tissue. For instance, let's say you are studying gene $X$, which is expressed in both brain and liver tissue. When you knock out gene $X$ using the standard approach, you find that the mice die as embryos due to a brain defect during embryogenesis. However, you are interested in the role of gene $X$ in the livers of adult mice. You can generate a conditional knockout of gene $X$ where it is only knocked out in adult livers but remains functional in embryonic brains so that they can be born and develop into adults that you can then study for liver function. One way to achieve this is to find a gene that is only expressed in adult livers, clone the upstream regulatory sequences that regulate expression in adult liver cells, and generate a transgene that expresses Cre under the control of this regulatory sequence. You can then generate a floxed allele (an allele with flanking $loxP$ sites) of gene $X$ and breed mice that carry this allele together with your liver-specific Cre transgene.
 ===== CRISPR/Cas9 =====
-As we learned in [[chapter_16|Chap. 16]], homologous recombination occurs only at low frequencies in mammalian cells – this is why it was necessary to develop technologies to culture ES cells in vitro, so that we could examine large numbers of ES cells to look for rare events. It turns out that the frequency of homologous recombination increases enormously if there is a double-stranded break in the DNA near the site you wish to have homologous recombination. CRISPR/Cas9 (Fig. {{ref>Fig4}} is an enzyme usually isolated from the bacterium //Streptococcus pyogenes// that can be used to cleave dsDNA to generate a double-stranded break. However, unlike restriction enzymes that only cut short defined palindromic sequences ([[chapter_09|Chap. 9]]), it is possible to modify CRISPR/Cas9 such that we can essentially make it cut almost any unique DNA sequence we wish. This is possible because CRISPR/Cas9 uses a guide RNA (gRNA) as part of its enzyme structure and the gRNA sequence is what guides it to its target DNA though RNA/DNA base pairing. It is relatively easy to modify this RNA sequence so that CRISPR/Cas9 will target the DNA sequence of a specific gene you are interested in. When dsDNA is cleaved in live cells, repair mechanisms will rejoin the cleaved DNA; however, errors often occur during this process, and indel mutations usually occur and therefore frameshifts, which usually creates null alleles. If a targeting construct is also present, homologous recombination will occur at a very high frequency; this allows for creation of knock-in alleles without necessarily using the Cre-$lox$-based strategy described above.
+As we learned in [[chapter_16|Chap. 16]], homologous recombination occurs only at low frequencies in mammalian cells – this is why it was necessary to develop technologies to culture ES cells in vitro, so that we could examine large numbers of ES cells to look for rare events. It turns out that the frequency of homologous recombination increases enormously if there is a double-stranded break in the DNA near the site you wish to have homologous recombination. CRISPR/Cas9 (Fig. {{ref>Fig4}}) is an enzyme usually isolated from the bacterium //Streptococcus pyogenes// that can be used to cleave dsDNA to generate a double-stranded break. However, unlike restriction enzymes that only cut short defined palindromic sequences ([[chapter_09|Chap. 9]]), it is possible to modify CRISPR/Cas9 such that we can essentially make it cut almost any unique DNA sequence we wish. This is possible because CRISPR/Cas9 uses a guide RNA (gRNA) as part of its enzyme structure and the gRNA sequence is what guides it to its target DNA through RNA/DNA base pairing. It is relatively easy to modify this RNA sequence so that CRISPR/Cas9 will target the DNA sequence of a specific gene you are interested in. When dsDNA is cleaved in live cells, repair mechanisms will rejoin the cleaved DNA; however, errors often occur during this process, and indel mutations usually occur and therefore frameshifts, which usually creates null alleles. If a targeting construct is also present, homologous recombination will occur at a very high frequency; this allows for creation of knock-in alleles without necessarily using the Cre-$lox$-based strategy described above.
 <figure Fig4>
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 </figure>
-Naturally occurring AAVs are small nonpathogenic parvoviruses. The AAV genome is just under 5 kbp long and is made of ssDNA instead of the more typical dsDNA. The ends of the ssDNA genome form structures called inverted terminal repeats (ITRs; Fig. {{ref>Fig5}}) that are required for replicating and packaging viral ssDNAs into new viral capsids. AAVs cannot successfully replicate in cells by themselves because they depend on proteins provided by another kind of virus (usually an adenovirus or herpresvirus) to produce new AAV viral particles after infection. For instance, three proteins from adenovirus, E2A, E4, and VA, are required for AAV replication.
+Naturally occurring AAVs are small nonpathogenic parvoviruses. The AAV genome is just under 5 kbp long and is made of ssDNA instead of the more typical dsDNA. The ends of the ssDNA genome form structures called inverted terminal repeats (ITRs; Fig. {{ref>Fig5}}) that are required for replicating and packaging viral ssDNAs into new viral capsids. AAVs cannot successfully replicate in cells by themselves because they depend on proteins provided by another kind of virus (usually an adenovirus or herpesvirus) to produce new AAV viral particles after infection. For instance, three proteins from adenovirus, E2A, E4, and VA, are required for AAV replication.
-To produce recombinant AAVS (rAAVs; Fig. {{ref>Fig6}}) as a gene delivery vector for research or therapeutic purposes, you would need to construct three separate plasmids in vitro:
+To produce recombinant AAVs (rAAVs; Fig. {{ref>Fig6}}) as a gene delivery vector for research or therapeutic purposes, you would need to construct three separate plasmids in vitro:
   * A plasmid that expresses the Rep and Cap proteins that are normally encoded by the AAV genome (purple in Fig. {{ref>Fig6}});
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   * A third plasmid that expresses the essential E2A, E4, and VA genes from adenovirus (green in Fig. {{ref>Fig6}})
-You would then introduce all three plasmids into cultured cells (such as HEK293 cells, a human embryonic kidney cell line). The purple and green plasmids, together with the HEK293 host cell genome, provide all the proteins needed to replicate and package newly replicated viral ssDNAs. However, only DNA with ITRs will be packaged. This means that the plasmids containing AAV and adenoviruses will not be packaged (they won't even be replicated) - only the ssDNA with the ITRs, and therefore your trangene, will be packaged into new viral capsids. You can harvest the rAAVs from the media of the HEK293 cell culture.
+You would then introduce all three plasmids into cultured cells (such as HEK293 cells, a human embryonic kidney cell line). The purple and green plasmids, together with the HEK293 host cell genome, provide all the proteins needed to replicate and package newly replicated viral ssDNAs. However, only DNA with ITRs will be packaged. This means that the plasmids containing AAV and adenoviruses will not be packaged (they won't even be replicated) - only the ssDNA with the ITRs, and therefore your transgene, will be packaged into new viral capsids. You can harvest the rAAVs from the media of the HEK293 cell culture.
 <figure Fig6>
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 </columns>
 <caption>
-Some example of AAV serotypes (variants) with different tissue targeting specificity (tropism). Adapted from Naso et al. (2017) BioDrugs 31(4): 317-334 https://doi.org/10.1007%2Fs40259-017-0234-5. Liscensing: [[http:/creativecommons.org/licenses/by-nc/4.0/|CC BY-NC-4.0]].
+Some example of AAV serotypes (variants) with different tissue targeting specificity (tropism). Adapted from Naso et al. (2017) BioDrugs 31(4): 317-334 https://doi.org/10.1007%2Fs40259-017-0234-5. Licensing: [[http://creativecommons.org/licenses/by-nc/4.0/|CC BY-NC-4.0]].
 </caption>
 </table>