Rnai

Methods to elucidate gene function

Historically, studies to identify genes that function in a particular process involve forward genetics. One way to mutate genes using this process is to expose organisms to a mutagen (typically either a chemical or gamma radiation), randomly mutating the genome in many animals. We then screen these animals for defects in the process we wish to study – essentially, 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. It is also a time-consuming process to map or figure out exactly where a mutation occurred and to make sure no additional mutations exist. A powerful alternative to forward genetics is to decrease the expression of genes with RNA interference (RNAi).

RNAi is an endogenous cellular mechanism that is present in some organisms, including plants and worms. 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. Silencing genes in this way is also called reverse genetics because one can knock down the function of particular genes in a targeted way without mutating the 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 (Figure 1). 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 a protein complex called RISC (RNAi-induced silencing complex). One strand from the siRNA is destroyed after siRNA binding, leaving the other strand bound to Argonaute, the RISC complex RNase. 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 target mRNAs within the complementary sequence, thereby inhibiting, or silencing, gene function. siRNA-bound RISC can then search for and destroy additional complementary target transcripts.

Figure 1: RNAi pathway (Alberts Essential Cell Biology 3rd edition)
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 (Figure 1). 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 a protein complex called RISC (RNAi-induced silencing complex). One strand from the siRNA is destroyed after siRNA binding, leaving the other strand bound to Argonaute, the RISC complex RNase. 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 target mRNAs within the complementary sequence, thereby inhibiting, or silencing, gene function. siRNA-bound RISC can then search for and destroy additional complementary target transcripts.

Figure 1: RNAi pathway (Alberts Essential Cell Biology 3rd edition)

RNAi in C. elegans
The nematode C. elegans is used as a model organism – one that is commonly studied by biological researchers. The RNAi mechanism was discovered using these organisms. Amazingly, RNAi can be activated in C. elegans by simply feeding worms bacteria expressing dsRNA corresponding 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 through transcript (mRNA) degradation. In C. elegans the triggering of the RNAi mechanism can spread from cell to cell, eventually knocking down function of the gene in the entire body. The experiment performed in this lab demonstrates how RNAi can be used to determine the function of genes. It is also a vivid demonstration of how interfering with the function of one gene can dramatically affect the phenotype, or appearance, of an organism.

You will examine RNAi-mediated knockdown of dpy-13 function. To generate dpy-13(RNAi) worms, wild-type worms onto both OP50-seeded NGM plates (as a control), and onto plates seeded with RNAi “feeding strains” of bacteria designed to turn down expression of a specific gene (here, dpy-13) through the RNAi mechanism.

Each RNAi feeding strain contains a plasmid (L4440) containing a piece of DNA sequence from the gene it is designed to silence. The introns have been removed from the DNA (cDNA) to allow bacterial cells to produce the proper Eukaryotic product. This cDNA was cloned into a multi-cloning site (MCS) (Figure 2). These plasmids were transformed into a special strain of E. coli with IPTG-inducible T7 polymerase activity to make bacterial RNAi feeding strains. The RNAi feeding strains must be grown on NGM/+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 promoters 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, allowing or inducing transcription of the target gene.

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. Again, we will knockdown dpy-13 function.

Figure 2: L4440 map (http://www.addgene.org). Note the amp resistance gene, Ampr, and each T7 promoter flanking the region where the gene of interest is inserted (MCS). Each T7 promoter directs transcription of a different strand of the DNA inserted into the MCS. dpy-13(e458) is a mutant, autosomal recessive allele of the dpy-13 gene that causes the worms to exhibit a dumpy (Dpy) phenotype when present in two copies. Last week you assessed the phenotype of both wild type (WT) and dpy-13(e458) worms using a microscope. This week, you will also examine dpy-13(RNAi) worms and you will predict the genotypes of WT, dpy-13(e458), and dpy-13(RNAi) worms. You will then test your prediction by genotyping each population of worms using PCR to determine whether each population contains the wild type or mutant dpy-5 allele. Since the dpy-13(e458) mutation is autosomal recessive, you will be able to predict whether the mutant worms are homozygous mutant, heterozygous or homozygous wild type. If the worms are heterozygous they should contain both a mutant copy and a wild type copy of the dpy-13 gene.

You should review the principles of PCR independently as this topic has been covered in your prerequisite courses. You will use PCR to amplify DNA from endogenous dpy-13 loci. The primers that you will use in your reactions are listed below:

5’-AGTCGTCTTCTCCGTTATCG-3’ (Forward Primer)
5’-GAGCAACGCATAAGGCAAAG-3’ (Reverse Primer)

These primers were designed to allow you to amplify DNA from both wild type and mutant dpy-13 alleles. You will perform PCR of whole worms to distinguish between worms that have the dpy-13 mutation and ones that do not. You will use BLAST to find the DNA sequence of the WT dpy-13 gene in the exercise below. The dpy-13(e458) mutation is a 673bp deletion of a part of the wild type DNA sequence. This deletion ends precisely at the TAA termination codon.

Complete the BLAST exercise and answer the questions that follow (Due Friday April 19th)

Use BLAST to Find DNA Sequences in Databases (Electronic PCR)
1. 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 (located under “Popular Resources”). Note: the location of BLAST on this Web page is sometimes altered.
c. Click on the link to nucleotide blast (blastn) under the heading Basic BLAST.
d. Enter the sequences of the primers into the Search window. These are the query sequences.
e. Omit any non-nucleotide characters from the window, because they will not be recognized by the BLAST algorithm.
f. Under Choose Search Set, select the Nucleotide collection(nr/nt) database from the drop-down menu.
g. Under Program Selection, optimize for somewhat similar sequences by selecting blastn.
h. 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 will attempt to match the primer sequences to the millions of DNA sequences stored in the database. While searching, a page showing the status of your search will be displayed 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.
2. The results of the BLAST search are displayed in three ways as you scroll down the returned page:
a. First, a graphical overview illustrates how significant matches, or “hits,” align with the query sequence.
b. This is followed by a table of significant alignments with links to database reports.
c. Next is a detailed view of each primer sequence (query) aligned to the nucleotide sequence of the search hit (subject). Notice that some hits have matches to both the forward primer (nucleotides 1–20) and to the reverse primer (21–40). Other hits are incomplete matches. Which of the hits would be amplified, in vitro, in a PCR reaction using the two primers? Why?
d. What do you expect would happen if you performed a BLAST search with only one primer?

3. Examine the hits from the BLAST search. What is the predicted length of the product that the primer set would amplify in a PCR reaction (in vitro)?
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. Shorter queries, such as primers, yield higher E-values.
b. Note the names of any significant alignments that have E-values less than 1. Do they make sense?
c. Scroll down to the Alignments section to see exactly where the two primers match a subject sequence.
d. The lowest and highest nucleotide positions in the subject sequence (Sbjct) indicate the borders of the amplified sequence. Subtracting one from the other gives the difference between the two coordinates.
e. However, the actual length of the fragment includes both ends, so add 1 nucleotide to the result to determine the exact length of the PCR product amplified by the two primers.
f. Repeat this calculation for at least three hits. Do you notice any discrepancy between the calculations? If so, why?

4. Click on the database accession link marked M23559.1 for the hit sequence). This opens a sequence report with three parts.
a. The top of the report has basic information about the sequence, including its base-pair length, database accession number, source, and references.
b. The middle section contains annotations of gene and regulatory features, listing the first and last nucleotide position of the feature.
• According to the features list, how many exons and introns does the dpy-13 gene have?
• What feature(s) are found between the nucleotide positions identified by the primers, as determined in step 3?
c. The bottom section of the report lists the entire nucleotide sequence of the gene or DNA sequence that contains the PCR product.
• Copy all the nucleotides between the beginning of the forward primer and end of the reverse primer.
• Paste this sequence into a text document. This is the amplicon, or amplified product. Turn in this sequence with the questions below.

Answer the following questions (Due Friday April 18th)

1. Given that the dpy-13(e458) mutation is a 673bp deletion of a part of the wild type dpy-13 DNA sequence, and that dpy-13(e458) behaves in an autosomal recessive manner, calculate the size of the expected PCR product/s amplified from WT, dpy-13(e458), and dpy-13(RNAi) worms. Fill in the table below.

Worms | Homozygous or heterozygous at dpy locus? | Predicted size of PCR product or products | WT | | | dpy-13(e458) | | | dpy-13(RNAi) | | |

2. What size PCR product/s would you observe if you performed this PCR reaction on a dpy-13(e458) heterozygote (dpy-13(e458)/+)?
3. You will analyze your PCR product using gel electrophoresis. Draw your gel below. Assume you are using a 100 bp ladder as a DNA marker. Indicate what you will load into each well of the gel. Indicate the size of the bands and placement of the electrodes relative to the gel (draw a + or – to indicate either red or black electrodes).
4. You will use PCR beads in your PCR reactions. These beads contain the reagents necessary to run the reactions. If you were to set up a PCR reaction from scratch, what would you need to add to the reaction? You may need to perform an internet search to answer this question.
Procedures:
Culturing C. elegans and RNAi feeding (This was already performed for you):
1. Use a black pen to label the bottom of an OP50-seeded plate with the date and “wild-type.”
2. Use a black pen to label the bottom of an OP50-seeded plate with the date and
“dpy-13(e458).”
3. Use a black pen to label the bottom of the plate seeded with the dpy- 13 RNAi feeding strain with the date and “wild-type.”
4. Pick five L4-stage worms (see illustration below) from your plate of wild-type worms to the OP50-seeded plate labeled “wild-type.”
5. Confirm that the transferred worms are the correct stage and were not injured or killed during the picking process.
6. Identify any eggs or young larvae that may have been accidentally transferred. Pick them off the plate, and flame them in a Bunsen burner.
7. Use the same method to move five L4 wild-type worms to the plate seeded with the dpy-13 RNAi feeding strain.
8. Use the same method to move five L4 dpy-13 worms to the OP50- seeded plate labeled “dpy-13(e458).”
9. Incubate the plates upside down at 20°C. Choose a place where the plates will not be disturbed.
10. The day after transferring, check that your worms are still healthy. Note any dead worms and pick them off the plate.
11. On the third day after transferring, pick the remaining adult worms off the plate. This will prevent confusion when scoring the progeny.

Isolation of chromosomal DNA from adult wt and mutant C. elegans

1. Label three PCR tubes with your group number. Label one tube “W”(wild type), one “R” (RNAi), and one “D” (deletion mutant).
2. Use a micropipet with a fresh tip to add 10 μL of lysis buffer to each tube.
3. Observe the wild-type, dpy-13(e458), and dpy-13(RNAi) worms under a dissecting microscope. Note differences in the length and width of the worms.
4. Use a sterile toothpick to transfer four or five adult wild-type worms to the “W” tube. Swish the tip directly in the lysis buffer to dislodge the worms from the pick.
5. Observe the PCR tube under a stereomicroscope. Verify that at least three worms are alive and writhing in the buffer. If you have trouble seeing the worms, flick the tube or momentarily centrifuge the tube to ensure that the worms are at the bottom.
6. Pick four or five dpy-13(RNAi) worms to the “R” tube, and verify that at least three live worms are in the lysis buffer.
7. Pick four or five dpy-13(e458) worms to the “D” tube, and verify that at least three live worms are in the lysis buffer.
8. Fit each of your PCR tubes into one or two adaptors. PCR tubes are “nested” into sequentially larger adaptors: a 0.2-mL tube, within a 0.5- mL tube, within a 1.5-mL tube.
9. Place your tubes in a balanced configuration in a microcentrifuge, and spin for 5–10 seconds at full speed. This will pellet the worms.
10. Place your tubes in liquid nitrogen, on dry ice, or in a -80°C freezer until the liquid in the tubes is frozen solid. (Freeze-thawing cracks the tough outer cuticle of the worm.)
11. Place your PCR tubes, along with other student samples, in a thermal cycler* that has been programmed for one cycle of the following profile.
The profile may be linked to a 4°C hold program.

Incubating step: 65°C, 90 minutes
Boiling step: 95°C, 15 minutes

*The PCR machine is used as a hot block to help break open the worm and inactivate enzymes in this step – no DNA amplification (PCR reaction) occurs here.

Proteinase K in the lysis buffer digests protein in the cuticle and helps to liberate individual cells by digesting protein fibers of the extracellular matrix that bind cells together. It also inactivates cellular proteins, including DNases that interfere with PCR amplification. The near-boiling temperature lyses individual cells and inactivates the proteinase K.
12. Store your sample on ice or at –20°C until you are ready to continue with the next step.

Amplification of C. elegans DNA by PCR

1. Obtain three tubes containing Ready-To-Go™ PCR Beads. Label each tube with your group number. Label one tube “W” (wild-type), one “R” (RNAi), and one “D” (deletion mutant).
2. Use a micropipet with a fresh tip to add 22.5 μL of dpy-13 primer/loading dye mix to each tube. Allow the bead to dissolve for a minute or so.
3. Use a micropipet with a fresh tip to add 2.5 μL of worm DNA from Part III to the appropriate tubes.
a. Add the DNA directly into primer/loading dye mix.
b. Ensure that no worm DNA remains in the tip after pipetting.
c. Use a fresh tip for each reaction.
4. If your thermal cycler does not have a heated lid: Prior to thermal cycling, you must add a drop of mineral oil on top of your PCR reaction. Be careful not to touch the dropper tip to the tube or reaction, or the oil will be contaminated with your sample.
5. Store your samples on ice until your class is ready to begin thermal cycling.
6. Place your PCR tubes, along with other student samples, in a thermal cycler that has been programmed for 30 cycles of the following profile.

The profile may be linked to a 4°C hold program after the 30 cycles are completed.
Denaturing step: 94°C 30 seconds
Annealing step: 55°C 60 seconds
Extension step: 72°C 60 seconds

7. After cycling, store the amplified DNA on ice or at –20°C until you are ready to continue with gel electrophoresis.
Analyze PCR Products by Gel Electrophoresis
1. Seal the ends of the gel-casting tray.
2. Insert a well-forming comb at one end of the tray.
3. Pour 1% TAE Agarose solution containing SyBR safe into the tray to a depth that covers about 1/3 the height of the open teeth of the comb.
4. Allow the gel to solidify completely. This takes approximately 20 minutes.
5. Remove the tape or sealing device, and place the gel into the electrophoresis chamber.
6. Add enough 1× TAE buffer to just cover the surface of the gel.
7. Carefully remove the comb, and add additional 1× TAE buffer to just cover and fill in wells, creating a smooth buffer surface. (Too much buffer increases running time by conducting electrical current over the gel.)
8. Use a micropipette with a fresh tip to load 20 μL of pBR322/BstNI size marker into the far left lane of the gel. (Alternatively, use 0.5 μg of 100-bp ladder, instructor will provide the ladder details on a day of the lab.)
9. Use a micropipette with a fresh tip to add 20 μL of each sample/loading dye mixture into a different lane of the gel. Draw a diagram first, so you can interpret your gel later. (If you used mineral oil during PCR, pierce your pipet tip through the layer of mineral oil to withdraw the PCR sample and leave the mineral oil behind in the original tube.)
10. Run the gel at 130 V for approximately 30 minutes. Adequate separation will have occurred when the cresol red and bromophenol blue dye front has moved at least 50 mm from the wells. Do not run the dye front to the end of the gel.
11. View the gel using a transilluminator, and image it using the LiCOR.

References
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Nature 391:806-811.
McMahon, L., Muriel, J.M., Roberts, B., Quinn, M., and Johnstone, I.L. (2003). Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix. Mol Biol Cell 14, 1366-1378.
Timmons, L., Court, D.L., and Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene.
263(1-2):103-112.

Results and Discussion 1. Starting 4 days after induction of RNAi by feeding, carefully examine each plate.
a. Have any eggs (i.e., embryos) been laid? Have any eggs hatched? If so, are the worms at a larval or an 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. Calculate the percentage of F1 adult worms that were affected on each RNAi plate. 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 and IPTG.
a. Why is ampicillin added to these plates?

b. Why is IPTG added to these plates?

c. How are the RNAi feeding strains of bacteria different from the OP50 strain, which is grown on plain NGM-lite plates?

d. Bacteria contain RNases. Why do you think the dsRNA is able to survive in the special strain of bacteria used to generate the RNAi feeding strain?

References: Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. McMahon, L., Muriel, J.M., Roberts, B., Quinn, M., and Johnstone, I.L. (2003). Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix. Mol Biol Cell 14, 1366-1378. Timmons, L., Court, D.L., and Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans