Studies to identify the function of a gene typically rely on mutating a particular gene, then looking for physical or behavioral changes in the organism.
Mutagenesis (creating mutations) is very time-consuming and generally cannot target specific genes, making it very difficult to study the function of a particular gene if no mutation in the gene already exists. A powerful alternative is to decrease the expression of genes with RNA interference (RNAi).
Biologists have learned to use the RNAi mechanism to their advantage. By deliberately introducing defined sequences of dsRNA identical to the sequence of a gene, biologists can observe the physiological consequences of
“silencing” that gene.
RNAi is a cellular mechanism thought to have evolved to protect organisms from infection by RNA viruses.
When double-stranded RNA (dsRNA) is present in a cell, it is recognized by a protein named Dicer. Dicer is an
RNase that cuts these dsRNA molecules into short pieces. These 21- to 25-base pair pieces, called small interfering
RNAs (siRNAs), are bound by another protein, called Argonaute. One strand from the siRNA is destroyed after siRNA binding, leaving the other strand bound to Argonaute. The remaining strand acts as a guide that hybridizes to messenger RNAs (mRNAs) that are complementary to the guide strand. Once bound, Argonaute cleaves the mRNAs within the complementary sequence, thereby inhibiting, or silencing, gene function.
The nematode C. elegans is one of the organisms commonly studied by biological researchers. C. elegans is a microscopic roundworm. Although some roundworms are parasitic, C. elegans is a free-living worm that feeds on soil bacteria. These worms grow quickly, developing from embryo to adult in 3 days. C. elegans is a simple animal with only about 1000 cells. Scientists know exactly how each of those cells develops from the fertilized egg. C.
elegans was the first multicellular organism to have its entire genome sequenced, with the surprising finding that
40% of its genes have human matches. Because of this, worms are a good system to study the biology of diseaseassociated genes. It is easier to control mating, isolate genes, and introduce foreign DNA in C. elegans than in more complex animals. All of these features make C. elegans a great model for understanding how cells divide, develop, and take on specialized tasks in higher (eukaryotic) organisms.
Amazingly, RNAi can be activated in C. elegans by simply feeding worms bacteria expressing dsRNA that corresponds to part of the gene to be silenced. The dsRNA enters cells through the intestine and is recognized by the
RNAi machinery, leading to silencing of the specific gene. In C. elegans the triggering of the RNAi mechanism can spread from cell to cell, eventually turning off the selected gene in the entire body.
The experiment performed in this lab demonstrates how RNAi can be used to figure out the function of genes. It is also a vivid demonstration of how interfering with the function of one gene can dramatically affect the
Inducing RNAi by Feeding phenotype, or appearance, of an organism. In Part I, wild-type worms are grown on plates containing worm media (NGM-lite) seeded with a strain of E. coli (OP50). In Part II, these worms are transferred onto both OP50-seeded NGM-lite plates (as a control), and onto plates seeded with RNAi “feeding strains” of bacteria designed to turn down specific genes (dpy-11 and bli-1) through the RNAi mechanism.
Each RNAi feeding strain contains a plasmid with a piece of DNA containing sequence from the gene it is designed to silence. The RNAi feeding strains must be grown on NGM-lite/amp+IPTG plates. The ampicillin in the plates selects for bacteria containing the plasmid, which includes a gene that confers ampicillin resistance. The double-stranded RNA (dsRNA) needed for RNAi is created when transcripts from both the coding and non-coding strands of the gene sequence cloned into the plasmid hybridize to each other. The transcription of these two RNAs begins from promoters on either side of the gene sequence. These promotors specifically use T7 RNA polymerase to initiate transcription. The IPTG in the plates induces expression of T7 RNA polymerase in the feeding strains of bacteria, inducing transcription.
Eating the bacteria that contain the double-stranded RNA triggers RNAi in the worms and turns down or
“down regulates” the gene. You can deduce the normal function of the gene by examining any phenotypes that result.

In accompanying bioinformatics exercises, you will discover the function of the protein encoded by each silenced gene using the online C. elegans database, called Wormbase, In addition, you will explore the relatedness of genes in worms and humans using the Basic Local Alignment Search Tool (BLAST).
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Prelab:
1.
List 2 purposes (learning targets) for this experiment.
2.
List 5 reasons why C. elegans is a good experimental organism.
3.
Make a diagram that demonstrates the role of Dicer and Arogonaute in gene control.
4.
What 2 genes are contained in the plasmids that have been inserted into the bacterial feeding strains?
What are their roles?
5.
Make a reference list including definition and function of: NGM-lite, OP50, dpy-11, bli-1, amp, and
IPTG.
Day I. GROW E. coli OVERNIGHT CULTURES
1. Label a sterile 15-mL culture tube with your group name or number and “OP50.”
2. Use a bulb pipet to sterilely transfer 1 mL of Luria Burtani (LB) broth into the tube. a. Ensure that the culture tube cap is unsnapped to the “loose” position. b. Peel apart the sterile wrapper of the bulb pipet at the bulb end ensuring the tip remains sterile. c. Remove the LB bottle cap using the little finger of the hand holding the pipet bulb. Flame the mouth of the
LB bottle. d. Withdraw 1 mL of LB. Re-flame the mouth of the bottle, and replace the cap. e. Remove the cap of the labeled OP50 sterile 15-mL culture tube. Expel the LB into the tube, and replace the cap.
3. Inoculate the liquid culture with OP50: a. Partially peel open the loop wrapper at the end opposite the loop. b. Use the loop to scrape a visible cell mass from the OP50 slant. Immerse the loop end in the broth, and agitate it to dislodge the cell mass. Discard the loop in the container of bleach. c. Replace tube cap in the loose position.
4. Label 2 sterile 15-mL culture tubes with your group name or number, the date, and one of the RNAi feeding strains per tube:
“bli-1 RNAi”
“dpy-11 RNAi”
5. Use a bulb pipet to sterilely transfer 1 mL of LB/amp broth into each tube (as in step 2 above).
6. Inoculate the bli-1 and dpy-11 RNAi cultures with bacteria from the appropriate slant cultures following the procedure in step 3.
7. Incubate the tubes at 37°C for 24–48 hours.
8. Take time for responsible cleanup.

Day II. SEED NGM-LITE AND NGM-LITE/amp+IPTG PLATES WITH E. coli
1. Label the bottom of 2 NGM-lite plates with your group name or number, and the date with a black pen. Use a blue pen to label the bottom of the plates “OP50.”
2. Use a bulb pipet to sterilely seed each of the NGM-lite plates with OP50. a. As before, peel open the wrapper from the bulb end. b. Remove the cap from the OP50 overnight culture using the little finger of your hand holding the pipet bulb. If using a Bunsen burner, briefly flame the mouth of the OP50 overnight culture. c. Use the bulb pipet to withdraw 1 mL of overnight culture. d. Add 1 to 2 drops of culture to the center of the surface of each NGM-lite plates. The drops should occupy most of the plate surface, but should not touch the edges of the dish.
3. Use a black pen to label the bottom of 2 NGM-lite/amp+IPTG plates with your group name or number and the date. Use the blue pen to label one of the feeding strains per plate:
“bli-1 RNAi”
“dpy-11 RNAi”
4. Use a bulb pipet to sterilely transfer 1 or 2 drops of overnight culture of each feeding strain to the appropriate
NGM-lite/amp+IPTG plate, as in step 2 above.
5. Grow the seeded plates face-up at room temperature for 24–36 hours. The bacterial lawn should be confluent and dry before any worms are added.
6. Take time for responsible cleanup in the same manner as on Day 1.
Day III. TRANSFER C. elegans TO OP50-SEEDED NGM-LITE PLATES
To feed worm strains, it is easier to transfer a chunk of worm-filled agar from a well-grown plate to a new plate rather than pick individual worms.
1. Use a black permanent marker to label the bottom of your OP50- seeded plate with the date and “wild-type.”
2. Sterilize a metal spatula. Dip the end into the ethanol, and briefly pass it through a Bunsen flame to ignite the alcohol. Allow alcohol to burn off away from the Bunsen flame and ethanol; the implement will become too hot if left in the flame.
CAUTION Be extremely careful not to ignite the ethanol in the beaker. Do not panic if the ethanol is accidentally ignited. Cover the beaker with a petri dish lid or other non-flammable cover to cut off oxygen and rapidly extinguish the fire.
4. Use the sterilized spatula to cut a 1-cm (about 3/8 in.) square chunk of agar with worms from the wild-type starter plate.
5. Transfer the chunk to your OP50-seeded plate, and place it face down in the agar. (Placing the chunk face down allows worms to crawl quickly into the bacteria on the new plate.)
6. Incubate the plate lid side down at +20°C (68°F) for 2 to 3 days. Choose a place where the plate will not be disturbed or exposed to direct sunlight. This will give the newly transferred worms time to grow.
Day IV. INDUCE RNAi BY FEEDING
1. Use a black permanent marker to label the bottom of an OP50-seeded plate with your group number or name, the date, and “wild-type.”
2. Use a black permanent marker to label the bottom of each of your RNAi feeding plates (bli-1 and dpy-11) with the date and “wild-type.”
3. Using the rounded end of a sterile toothpick, pick a small blob of bacteria from the edge of the bacterial lawn of an unused OP50- seeded plate.
4. While observing the worms with a dissecting microscope, transfer five L4-stage worms from your plate of wildtype worms to the new OP50-seeded plate labeled “wild-type.” See step 8 for information about L4 larvae. Use the blob of E. coli on the end of your pick as glue to pick up the worms. If you have trouble identifying L4 larvae, pick medium-sized worms from the wild-type plate. You may have to pick the worms one at a time.

5. Confirm that the transferred worms were not injured or killed during the picking process.
6. Identify any eggs or young larvae that may have been accidentally transferred. Carefully pick them off the plate with a sterile toothpick and dispose of them appropriately.
7. Use the same method, using new sterile toothpicks for each plate, to transfer five L4 wild-type worms to each of the plates seeded with RNAi feeding strains (bli-1 and dpy-11).
8. Incubate the plates lid side down at +20°C (68°F). Choose a place where the plates will not be disturbed or exposed to direct sunlight.
The following images and diagram of the C. elegans life cycle will help identification of L4 hermaphrodites. In between each larval stage and after the L4 stage, worms molt. This enables them to continue growing. a. An adult hermaphrodite (a worm with both male and female gametes) is a large worm with embryos inside.
Hermaphrodites are self-fertile, but can also mate with males. The wild-type strain used in this experiment contains few if any adult males; wild-type C. elegans does not have females. b. The embryo is a small, oval object. c. An L1 larva has recently hatched and is the smallest of the four larval stages. d. L2 and L3 larvae are larger than L1 worms, but not as large as an adult. Examine worms of different sizes to familiarize yourself with these larval stages. e. The final juvenile stage, an L4 larva, is almost as large as an adult hermaphrodite. The lack of internal embryos is one marker that distinguishes an L4 larva from an adult. A clear, crescent-shaped patch near the center of the body is another characteristic of an L4 larva. The egg-laying structure, called the vulva, will develop in this patch when the L4 molts into an adult.
RESULTS AND DISCUSSION

1. Starting 2 days after induction of RNAi by feeding, carefully examine each plate. Organize this information into a computer-generated data table for submission. a. Have any eggs (i.e., embryos) been laid? Have any eggs hatched? If so, are the worms at a larval or adult stage? b. Compare the offspring of the RNAi-treated worms with the offspring of the untreated wild-type worms.
Note any differences in morphology or behavior. At which developmental stage are the worms in which you observe these differences (L1, L2–3, L4, or adult)? c. Make a quantitative assessment of the RNAi effect on phenotype on each plate. Once some of the 1st generation (F1) progeny of the RNAi-treated worms reach adulthood, look for any worms on the plate that have an unusual phenotype. d. Count the number of adult F1 worms that have an unusual phenotype and those that appear normal
(wild type). As you count each worm, remove it from the plate with a toothpick. e. Repeat this analysis again the following day for additional F1 worms that have reached adulthood. f. Calculate the percentage of F1 adult worms that were affected on each RNAi plate on each day. How effective was the RNAi treatment? Did the RNAi effect vary over time?
2. Given the phenotypes you observed, what can you deduce about the function of each gene that had its expression decreased?
3. NGM-lite plates on which the RNAi feeding strains are grown include ampicillin. Why is ampicillin added to these plates? How are the RNAi feeding strains of bacteria different from the OP50 strain, which is grown on
plain NGM-lite plates?
BIOINFORMATICS
For a better understanding of the experiment, do the following bioinformatics exercises before you analyze your results.
Information is encoded in the nucleotide sequence of DNA. Bioinformatics is the field that identifies biological information in DNA using computer-based tools and algorithms. Algorithms are step-by-step procedures for solving a problem. Some bioinformatics algorithms aid in the identification of genes, promoters, and other functional elements of
DNA. Other algorithms help determine the evolutionary relationship between DNA sequences.
Because of the large number of tools and DNA sequences available, experiments done in silico (“in silicon,” or on the computer) now complement experiments done in vitro (“in glass,” or in a test tube). This movement between molecular biology and computation is a key feature of modern biological research.
In Part I, you will use Wormbase, an online database for C. elegans research, to learn about one of the genes examined in the laboratory. Wormbase contains the entire C. elegans genome sequence and the locations of all predicted genes. The entry for each gene includes a summary of data from genetic, biochemical, and RNAi experiments. In Part II, you will use the Basic Local Alignment Search Tool (BLAST) to identify the human equivalent of this same worm gene. This approach can be repeated with the other gene used in the laboratory.
I. Use Wormbase to Find Basic Information About a Gene
1. Open Wormbase at http://www.wormbase.org/.
2. Enter “dpy-11” in the text box adjacent to Search: for a Gene, and then click the search button.
3. Look at the description of the gene. What does “dpy” stand for? What sort of protein does the gene encode?
What is the function of the gene? Follow any blue links to find additional information.
4. Scroll down to the Location section to examine a graphical representation of the structure of the dpy-11 gene on C.
elegans chromosome V. You may need to select “download” HTML format. Parts of two neighboring genes are also depicted. Three dpy-11 transcripts are depicted, each running from right to left, or 5’ to 3’. Exons are indicated by colored boxes, introns by thin black lines.
5. Scroll down to the Function section, which describes different alleles (mutations) of the dpy-11 gene. If you do not see a table with two columns labeled “Phenotypes” and “Supporting Evidence,” click on the heading “Phenotype

Summary.” Each allele is given a different number. In this laboratory, you used dpy-11(e794), allele e794 of the dpy-
11 gene. In the Genetics section, follow the blue link for a complete description of this allele.
II. Use BLAST to Find the Human Homolog of dpy-11
1. Return to the Identification section at the top of the page, and find the Gene model(s) table. Click on the second entry in the Nucleotides (coding/transcripts) column, to retrieve the nucleotide sequence for the transcript
“F46E10.9.2.”
2. In the new window, the first sequence is the spliced coding region (transcript) as it would be translated by the ribosome. (For simplicity’s sake, the transcript is presented as DNA code, rather than RNA.) Exons are shaded in alternating yellow and orange (exon 1 is yellow, exon 2 is orange, exon 3 is yellow, etc.). The second sequence is the unspliced gene. Exons are colored, while introns and untranslated regions are white.
3. Copy the entire spliced sequence; you will paste it in a subsequent step.
4. Initiate a BLAST (Basic Local Alignment Search Tool) search. a. Open the Internet site of the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/ . b. Click on BLAST. c. Click on blastx to select a search using a protein database and translated nucleotide query. d. Paste the dpy-11 coding sequence into the Search window. e. Delete any non-nucleotide characters from the window. f. In the Choose Search Set section, type “Homo sapiens” in the Organism window. g. Click on BLAST! and the query sequences are sent to a server at the National Center for Biotechnology
Information in Bethesda, Maryland. There, the BLAST algorithm, or program, will translate the nucleotide sequence and attempt to match the amino acid sequence to the millions of protein sequences stored in its database. A page shows the status of your search until your results are available. This may take only a few seconds, or more than a minute if a lot of other searches are queued at the server.
5. The results of the BLAST search are displayed in three ways on the output page: a. A graphical overview illustrates how significant matches (also known as “hits”) align with the query sequence. b. A list of significant alignments with Accession links. c. A detailed view of the translated dpy-11 sequence (query) aligned to the amino acid sequence of each search hit (subject). Amino acids that are identical to both sequences are listed in the center row, while biochemically similar amino acids are indicated by “+” signs.
6. Examine the hits from the BLAST search. a. In the list of significant alignments, notice the scores in the E-value column on the right. The Expectation or
E-value is the number of alignments with the query sequence that would be expected to occur by chance in the database. The lower the E-value, the higher the probability that the hit is related to the query. Longer queries generally yield lower E-values. b. An alignment is considered significant if it has an E-value less than 0.1. Consider that the value 2e-55 denotes 2 × 10–55, or a 2 preceded by 54 decimal places! c. Identify the hits with the lowest E-values. Hits with similar E-values are usually variants of the same sequence. What human protein is most related to C. elegans DPY-11? What E-value did it yield? Is it a significant match? d. Find a hit with a dark “G” next to the entry and click on the “G.” This link will open an Entrez Gene record for the human dpy-11 relative. e. From the Display drop-down menu at the top left, select Full Report. Scroll down, and notice that the report includes a graphic representation of transcripts for the gene, its chromosomal location, and a link to
Related Articles in Pubmed. Does this gene have a similar function to dpy-11? Is it important in human health? f. Follow any of the links to get more detailed information about the gene.