These labs were modified from the work of Cassandra Theusch at the University of Wisconsin. We are grateful to her for sharing this curriculum.
Lab 6: Obtain CL2070 worms and propagate
Lab 7: Examination of CL2070 hsp-16.2::GFP worms
Lab 8: Creating the feeding strain of bacteria for RNAi
Lab 9: Induction of feeding strain to produce dsRNA
Lab 10: Phenotypic analysis of treated vs untreated worms
Lab 11: Optional Writing Conferences
Series 3: Background Information on Reverse Genetics: Using RNAi and Gene Regulation to Investigate Gene function 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, we use the powerful tool of interference RNA, RNAi--- a way to silence or downregulate a gene in order to study its function. In this reverse genetics study we will start with a known normal gene rather than a mutant phenotype and we hope to prevent the normal gene from producing its product. Our overall goal is to discover the function of that gene and gene product directly and, perhaps. to find out whether or not it 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.
- Your instructor will construct or obtain a plasmid containing the gene of interest
- You will isolate that plasmid from the cloning strain of bacteria
- You will transform the plasmids (with gene interest) into HT115 (DE3) E. coli baterial cells genetically modified to have impaired ability to degrade double stranded RNA
- You will select for transformants on media with ampicillin
- You will choose an isolated colony to culture and make lots of feeder strain bacteria
- You will induce expression of C. elegans gene dsRNA from the RNAi plasmid in the bacteria by IPTG induction
- You will seed special worm growth media plates with feeder strain bacteria
B. You will add worms to the plates of this special feeder strain of bacteria (bacteria expressing dsRNA of our gene of interest). Ingesting this gene specific dsRNA should cause the nematodes to show reduced or "knockdown" expression of our gene of interest. We will use wild type C. elegans worms (N2 and rrf-3 strains) and CL2070 hsp-16.2::GFP worms.
C. You will observe and compare phenotype differences among the progeny of the three strains of worms that were fed the special RNAi feeder strain and those fed normal OP50 bacteria.
Schedule of Experiments
||Outside lab time
||Begin a culture of CL2070 worms
||10/22 - 10/28
||Examine the CL2070 strain of worms
||11/4 - 11/8
||Transform hsf-1 feeding RNAi plasmid into the HT115DE3 strain
||The day after lab: check your transformed bacteria for colony growth|
The night before next lab: set up an overnight culture of your colony of interest
||11/11 - 11/15
||Induction of dsRNA production, seeding of RNAi plates
||Add different strains of worms to feeding plates at appropriate time
||11/18 - 11/22
||Students score N2, rrf-3 worms for phenotypes and examine the CL2070 worms under the fluorescent scope
||11/26 - 11/30
||Individual conferences as needed