BISC219/F12: RNAi General Information

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (11:00, 22 August 2012) (view source)
(Outline of Experimental Design for Project 3: Investigating Gene Regulation Using RNAi)
 
(3 intermediate revisions not shown.)
Line 3: Line 3:
''
''
==Series 3: Background Information on Investigating Gene Regulation using RNAi in ''C. elegans''==
==Series 3: Background Information on Investigating Gene Regulation using RNAi in ''C. elegans''==
-
In forward or classical 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 that investigates gene regulation using RNAi, we will start with a known normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product. We hope to discover the function of that gene and gene product directly and, perhaps. to find out whether or not is has a regulatory function on other genes.  
+
In forward or classical 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 that investigates gene regulation using RNAi, we will start with a known normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product. We hope to discover the function of that gene and gene product directly and, perhaps. to find out whether or not is has a regulatory function on other genes.  
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 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.  
Line 25: Line 25:
== Outline of Experimental Design for Project 3: Investigating Gene Regulation Using RNAi ==
== Outline of Experimental Design for Project 3: Investigating Gene Regulation Using RNAi ==
Keep in mind that you will not do all of these steps yourself but you should understand why and how they were done and, at all times, be able to answer these questions: '''Where are you now in this process?'''  (What have you done so far? What's next?)<BR>
Keep in mind that you will not do all of these steps yourself but you should understand why and how they were done and, at all times, be able to answer these questions: '''Where are you now in this process?'''  (What have you done so far? What's next?)<BR>
-
'''A.'''  Make the feeder strain of bacteria that has a gene of interested ''Knocked Out'' or ''Knocked Down''<BR>
+
'''A.'''  Make the feeder strain of bacteria that expresses double stranded RNA from our gene of interest.<BR>
-
# Amplify gene of interest by PCR <BR>
+
# Your instructor will select for bacteria containing a plasmid containing the gene of interest on media with a selectable marker  
-
# Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation<BR>
+
# You will culture a selected colony to create a lot of copies of these bacteria
-
# Clean up DNA (remove enzymes) <BR>
+
# You will isolate the cloned plasmid DNA from that cultured colony by miniprep
-
# Cloning: ligate gene into vector plasmid with amp resistance gene <BR>
+
# You will transform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade double stranded RNA
-
# Transform competent bacterial cells
+
# You will select for transformants on media with ampicillin  
-
# Select for transformants on media with a selectable marker <BR>
+
# You will choose an isolated colony to culture and make lots of feeder strain bacteria  
-
# Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interest
+
# You will induce expression of ''C. elegans'' gene dsRNA from the pL4440 vector in the bacteria by IPTG induction  
-
# Culture the selected colony from colony pcr to create a lot of copies of these bacteria
+
# You will seed special worm growth media plates with feeder strain bacteria
-
# Isolate the cloned plasmid DNA from that cultured colony by miniprep<BR>
+
# You will add worms to this media and let them grow to allow for "knockdown" of the expression of our gene of interest
-
# Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA<BR>
+
'''B.'''  Add phenotypically wild type ''C. elegans'' worms (N2 and ''rrf-3'' strains) on the feeding plates that containing bacteria expressing dsRNA of our gene of interest (feeder strain of bacteria). <BR>
-
# Select for transformants on media with ampicillin  
+
-
# 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 special worm growth media plates and chemotaxis plates with feeder strain <BR>
+
-
'''B.'''  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>
+
'''C.'''  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>
'''C.'''  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>
 +
==Links to Labs& Project Info==
 +
'''Series1:'''<BR>
 +
[[BISC219/F12: Worm Info | Worm Info]] <br>
 +
[[BISC219/F12: Gene Linkage | Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start]]<BR>
 +
[[BISC219/F12: Lab 2 | Lab 2: Sex-Linked or Autosomal Finale]]<br>
 +
'''Series2:'''<BR>
 +
[[BISC219/F12: Gene Mapping Info | Background: Classical Forward Genetics and Gene Mapping]]<br>
 +
[[BISC219/F12: Lab 2 Mutant Hunt | Lab 2: Mutant Hunt]]<br>
 +
[[BISC219/F12: Lab 3  | Lab 3: Linkage Test Part 1]]<br>
 +
[[BISC219/F12: Lab 4  | Lab 4: Linkage Test Part 2, Mapping and Complementation]]<br>
 +
[[BISC219/F12: Lab 5  | Lab 5: Finish Complementation; Mapping Continued]]<br>
 +
[[BISC219/F12: Lab 6 | Lab 6: DNA sequence analysis; Mapping Continued]]<BR>
 +
[[BISC219/F12: Lab 7  | Lab 7: Complete Mapping: Score]]<br>
 +
'''Series3:'''<BR>
 +
[[BISC219/F12: RNAi General Information| Background Information on Project 3: Investigating Gene Regulation Using RNAi]] <br>
 +
[[BISC219/F12: Media Recipes | Media Recipes]]<br>
 +
[[BISC219/F12: RNAi Lab 7  | Lab 7: Identifying a bacterial colony containing our plasmid of interest  ]]<br>
 +
[[BISC219/F12: RNAi Lab 8  | Lab 8: Creating the feeding strain of bacteria for RNAi]]<br>
 +
[[BISC219/F12: RNAi Lab 9  | Lab 9: Induction of feeding strain to produce dsRNA and feeding worms]]<br>
 +
[[BISC219/F12: RNAi Lab 10 | Lab 10: Phenotypic analysis of treated vs untreated worms]]<br>
 +
[[BISC219/F12: RNAi Lab 11 | Lab 11: Writing Workshop]]<br>
 +
[[BISC219/F12: RNAi Lab 12 | Lab 12: Writing Conferences]]<br>
 +
 +
</div>

Current revision

Series 3: Background Information on Investigating Gene Regulation using RNAi in C. elegans

In forward or classical 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 that investigates gene regulation using RNAi, we will start with a known normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product. We hope to discover the function of that gene and gene product directly and, perhaps. to find out whether or not is has a regulatory function on other genes.

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 Project 3: Investigating Gene Regulation Using RNAi

Keep in mind that you will not do all of these steps yourself but you should understand why and how they were done and, at all times, be able to answer these questions: Where are you now in this process? (What have you done so far? What's next?)
A. Make the feeder strain of bacteria that expresses double stranded RNA from our gene of interest.

  1. Your instructor will select for bacteria containing a plasmid containing the gene of interest on media with a selectable marker
  2. You will culture a selected colony to create a lot of copies of these bacteria
  3. You will isolate the cloned plasmid DNA from that cultured colony by miniprep
  4. You will transform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade double stranded RNA
  5. You will select for transformants on media with ampicillin
  6. You will choose an isolated colony to culture and make lots of feeder strain bacteria
  7. You will induce expression of C. elegans gene dsRNA from the pL4440 vector in the bacteria by IPTG induction
  8. You will seed special worm growth media plates with feeder strain bacteria
  9. You will add worms to this media and let them grow to allow for "knockdown" of the expression of our gene of interest

B. Add phenotypically wild type C. elegans worms (N2 and rrf-3 strains) on the feeding plates that containing bacteria expressing dsRNA of our gene of interest (feeder strain of bacteria).
C. 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.

Links to Labs& Project Info

Series1:
Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Series2:
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Continued
Lab 6: DNA sequence analysis; Mapping Continued
Lab 7: Complete Mapping: Score
Series3:
Background Information on Project 3: Investigating Gene Regulation Using RNAi
Media Recipes
Lab 7: Identifying a bacterial colony containing our plasmid of interest
Lab 8: Creating the feeding strain of bacteria for RNAi
Lab 9: Induction of feeding strain to produce dsRNA and feeding worms
Lab 10: Phenotypic analysis of treated vs untreated worms
Lab 11: Writing Workshop
Lab 12: Writing Conferences

Personal tools