BISC 219/F10:RNAi General Information

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(Outline of Experimental Design for REVERSE Genetics Project)
Current revision (09:55, 18 May 2011) (view source)
 
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==Series 3: Reverse Genetics using RNAi in ''C. elegans''==
==Series 3: Reverse Genetics using RNAi in ''C. elegans''==
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In forward genetics, a mutant phenotype is attributed to one or more mutations in the DNA sequence of a gene. One disadvantage to Forward Genetics projects such as ours is that we studied a defective gene to find out how its product was functionally defective and then we had to infer the function of the normal form of the gene product. Ultimately, we care most about what that mutation can tell us about normal gene function. In our new reverse genetics projects, we will start with a normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product and discover the function of that gene and gene product directly. In the past, gene disruption (the starting point in reverse genetics studies) of a normal gene was done in many different ways. One of the most common was by creating a "knockout": an organism in which the gene of interest was deleted, and thus "silenced", but the organism was still viable for study. Most commonly, this was done in mice. These knockout mice were VERY expensive and difficult to produce and maintain and a limited number of genes could be studied.  
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In forward genetics, a mutant phenotype is attributed to one or more mutations in the DNA sequence of a gene. One disadvantage to Forward Genetics projects, such as ours, is that we studied a defective gene to find out how its product was functionally defective and then we had to infer the function of the normal form of the gene product. Ultimately, we care most about what that mutation can tell us about normal gene function. In our new reverse genetics projects, we will start with a normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product and discover the function of that gene and gene product directly. In the past, gene disruption (the starting point in reverse genetics studies) of a normal gene was done in many different ways. One of the most common was by creating a "knockout": an organism in which the gene of interest was deleted, and thus "silenced", but the organism was still viable for study. Most commonly, this was done in mice. These knockout mice were VERY expensive and difficult to produce and maintain and a limited number of genes could be studied.  
In recent years, the usefulness of the ''C. elegans'' model system in reverse genetic analysis of normal genes has been dramatically enhanced because this organism is particularly suited to gene silencing by RNA interference (RNAi).  RNAi disrupts gene expression in an entirely different way than knocking out a gene by removing it from the genome – RNAi works by targeting specific mRNA transcripts for destruction. RNAi is a mechanism that inhibits gene function when double-stranded RNA (dsRNA) molecules that correspond to part of a “target gene” are present in a cell.  By deliberately introducing defined sequences of dsRNA, biologists can observe the physiological consequences of “silencing” virtually any gene in ''C. elegans'', as well as many other plants and animals.<BR>
In recent years, the usefulness of the ''C. elegans'' model system in reverse genetic analysis of normal genes has been dramatically enhanced because this organism is particularly suited to gene silencing by RNA interference (RNAi).  RNAi disrupts gene expression in an entirely different way than knocking out a gene by removing it from the genome – RNAi works by targeting specific mRNA transcripts for destruction. RNAi is a mechanism that inhibits gene function when double-stranded RNA (dsRNA) molecules that correspond to part of a “target gene” are present in a cell.  By deliberately introducing defined sequences of dsRNA, biologists can observe the physiological consequences of “silencing” virtually any gene in ''C. elegans'', as well as many other plants and animals.<BR>
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# Clean up DNA (remove enzymes); <BR>
# Clean up DNA (remove enzymes); <BR>
# Cloning: ligate gene into vector plasmid with amp resistance gene ;<BR>
# Cloning: ligate gene into vector plasmid with amp resistance gene ;<BR>
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# Transform competent bacterial cells of a strain genetically modified to be tetracycline resistant;  
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# Transform competent bacterial cells;  
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# Select for transformants on media with tetracycline and ampicillin;<BR>
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# Select for transformants on media with ampicillin;<BR>
# Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
# Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
# Culture the selected colony from colony pcr to create a lot of copies of these bacteria
# Culture the selected colony from colony pcr to create a lot of copies of these bacteria
# Isolate the cloned plasmid DNA from that cultured colony by miniprep;<BR>
# Isolate the cloned plasmid DNA from that cultured colony by miniprep;<BR>
# Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;<BR>
# Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;<BR>
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# Select for transformants on media with tetracycline and ampicillin  
+
# Select for transformants on media with ampicillin  
# Choose an isolated colony to culture and make lots of feeder strain bacteria; <br>
# Choose an isolated colony to culture and make lots of feeder strain bacteria; <br>
 +
# Induce expression of ''C. elegans'' gene dsRNA from the pL4440 vector in the bacteria by IPTG induction. <br>
# Seed NM lite worm growth media plates with feeder strain produced as described <BR>  
# Seed NM lite worm growth media plates with feeder strain produced as described <BR>  
Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>
Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>
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Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that we NOT fed feeder strain bacteria.<BR><BR>
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Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR>
Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>
Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>
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Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.<BR><BR>
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Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>
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Visualize cDNA in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of  coding regions of gene of interest.
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Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.
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<div class=noprint>
==Links to Labs& Project Info==
==Links to Labs& Project Info==
Series1:<BR>
Series1:<BR>
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[[BISC 219/F10: RNAi Lab 10 | Lab 10: Scoring your worms and RNA purification]]<br>
[[BISC 219/F10: RNAi Lab 10 | Lab 10: Scoring your worms and RNA purification]]<br>
[[BISC 219/F10: RNAi Lab 11 | Lab 11: RT PCR reactions]]<br><br>
[[BISC 219/F10: RNAi Lab 11 | Lab 11: RT PCR reactions]]<br><br>
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</div>

Current revision

Series 3: Reverse Genetics using RNAi in C. elegans

In forward genetics, a mutant phenotype is attributed to one or more mutations in the DNA sequence of a gene. One disadvantage to Forward Genetics projects, such as ours, is that we studied a defective gene to find out how its product was functionally defective and then we had to infer the function of the normal form of the gene product. Ultimately, we care most about what that mutation can tell us about normal gene function. In our new reverse genetics projects, we will start with a normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product and discover the function of that gene and gene product directly. In the past, gene disruption (the starting point in reverse genetics studies) of a normal gene was done in many different ways. One of the most common was by creating a "knockout": an organism in which the gene of interest was deleted, and thus "silenced", but the organism was still viable for study. Most commonly, this was done in mice. These knockout mice were VERY expensive and difficult to produce and maintain and a limited number of genes could be studied.

In recent years, the usefulness of the C. elegans model system in reverse genetic analysis of normal genes has been dramatically enhanced because this organism is particularly suited to gene silencing by RNA interference (RNAi). RNAi disrupts gene expression in an entirely different way than knocking out a gene by removing it from the genome – RNAi works by targeting specific mRNA transcripts for destruction. RNAi is a mechanism that inhibits gene function when double-stranded RNA (dsRNA) molecules that correspond to part of a “target gene” are present in a cell. By deliberately introducing defined sequences of dsRNA, biologists can observe the physiological consequences of “silencing” virtually any gene in C. elegans, as well as many other plants and animals.

Amazingly, this mechanism can be activated in C. elegans by simply feeding worms bacteria expressing dsRNA that corresponds to part of the gene to be silenced. An altered phenotype in the progeny of RNAi-treated worms indicates what happens when the normal function of this gene is lost. The other two methods of RNAi in C. elegans are the soaking method, in which animals are soaked in dsRNA, and the injection method, in which dsRNA is microinjected into worms.

How does this happen in the worms? The enzyme dicer recognizes dsRNA and degrades (or cuts) it into siRNA (small interfering RNA), which is then taken into the RISC complex that degrades mRNA sequences that are identical (or close to identical) to the siRNA. As a historical sidelight, although previously observed in a number of other organisms, RNAi was truly developed using C. elegans. This resulted in a 2006 Nobel Prize to Craig Mello at UMass Medical Center in Worcester, MA and Andy Fire at Carnegie Mellon, for their research in this area. This is one of 3 Nobel Prizes won using C. elegans as a model organism!

I could recreate the history of RNAi here to explain it but many more people have done it better than I ever could. Here is a link to a great overview of RNAi and its history from Ambion Biosciences RNAi Pages. Please examine The Overview of RNA interference and The Mechanism of RNA interference. This is a great beginning to understanding RNAi not only in non-mammalian cells but also the differences between non-mammalian and mammalian gene silencing.

You might also want to check out NOVA Science Now RNAi Explained.

Here is a link to animations from Nature: Animations.

Remember that the ultimate goal of both forward and reverse genetic analyses is essentially the same: to understand the importance of a gene. What does its product do in the model organism and in other species? The basic differences in reverse genetics compared to forward is in where we start (gene in reverse vs. gene product in forward). In our particular reverse genetics study, we will be able to silence a normal gene without disrupting the DNA.

Outline of Experimental Design for REVERSE Genetics Project

Where are you now in this process?(What have you done so far; What's next?)
Make the feeder strain of bacteria

  1. Amplify gene of interest by pcr ;
  2. Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
  3. Clean up DNA (remove enzymes);
  4. Cloning: ligate gene into vector plasmid with amp resistance gene ;
  5. Transform competent bacterial cells;
  6. Select for transformants on media with ampicillin;
  7. Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
  8. Culture the selected colony from colony pcr to create a lot of copies of these bacteria
  9. Isolate the cloned plasmid DNA from that cultured colony by miniprep;
  10. Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
  11. Select for transformants on media with ampicillin
  12. Choose an isolated colony to culture and make lots of feeder strain bacteria;
  13. Induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction.
  14. Seed NM lite worm growth media plates with feeder strain produced as described

Plate wild type C. elegans worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).

Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.

Isolate RNA from RNAi worms and control worms of same strains.

Perform RT-PCR (Reverse Transcriptase)to amplify the C. elegans gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.

Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.

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