Exploring Gene Function in C. elegans: Mutations and RNA Interference
Carolina Biological Supply Company
Agenda
• What are model organisms?
• Introduction to C. elegans
• Hands-on
• RNAi mechanism
- RNAi by feeding
• Hands-on
• Real-world connections
How do we determine the function of a gene?
Find a Tractable System
• Ease of cultivation
• Simplicity
• Relevant biology
• Ease of manipulation
“(W)hen I embarked on this problem, I decided that what was needed was an experimental organism which was suitable for genetical study and in which one could determine the complete structure of the nervous system.” S. Brenner, Genetics 77: 71−94, May 1974
What Makes a Good Model Organism?
Photo courtesy of Matthew Meselson
Profile Organism: Nematode (roundworm)
Location: Temperate soil
Food: Soil bacteria
Genome size: 100 Mb; sequenced
Number of chromosomes: 6
Somatic cells: 959; fully traced
Egg-adult time: 3 days
Lifespan: 2 to 3 weeks
Reproduction: 250 to 1,000 progeny
Our Model Organism: Caenorhabditis elegans
= ~
About 40% of worm genes are related to human genes.
Gut, muscle, skin, and nervous
systems are like ours.
Life Cycle of a Worm
C. elegans Phenotypes
bli-1 wild type dpy-11
Blistering of cuticle Shorter bodies
Students will:
• Examine worm behavior and morphology
• Identify stages of worm development
• Compare mutant strains and to wild type
• Practice culturing C. elegans
Examining, Growing, and Caring for Worms
Culturing and Observing C. elegans Kit Item #211390
Identify mutants
Deduce the function of mutated genes
How Do We Study Gene Function?
Function
Can We Go Directly from Sequence to Function?
RNAi
RNA Interference
Gene X mRNA No Protein
RNAi allows you to specifically destroy the mRNA from a single gene.
DNA (Gene) mRNA Protein
First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans, which led to an efficient sequence-specific silencing and coined the term "RNA interference”
Fire et al. 1998
Negative control Uninjected
mex-3B antisense RNA mex-3B dsRNA
Antisense RNA
Weak effect
dsRNA
Strong effect
Double-stranded RNA was a contaminant in antisense experiments.
dsRNA Halts Gene Function
Dicer Cuts dsRNA into Short RNAs
Nucleus
dsRNA
2001
Bernstein et al.
Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as 2 dsRNA-binding domains
Dicer
Dicer Cuts dsRNA into Short RNAs
Nucleus
dsRNA
2001
Bernstein et al.
Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as 2 dsRNA-binding domains
Dicer
Dicer Cuts dsRNA into Short RNAs
Nucleus
2001
Bernstein et al.
Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as 2 dsRNA-binding domains
Dicer siRNA
RISC Uses siRNAs to Slice Transcripts
Nucleus
RISC (RNA-inducing
silencing complex)
siRNA
2004
Song et al.
Solved the crystal structure of pyrococcus Argonaute, showing it is slicer
RISC Uses siRNAs to Slice Transcripts
Nucleus
RISC (RNA-inducing
silencing complex)
siRNA
RISC Uses siRNAs to Slice Transcripts
Nucleus
RISC (RNA-inducing
silencing complex)
RISC Uses siRNAs to Slice Transcripts
Nucleus
RISC (RNA-inducing
silencing complex)
RISC Uses siRNAs to Slice Transcripts
Nucleus
RISC (RNA-inducing
silencing complex)
RISC Uses siRNAs to Slice Transcripts
Nucleus
RISC (RNA-inducing
silencing complex)
Gene function stopped
Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998)
C. elegans, Methods for RNAi
This activity uses RNAi by feeding
Bacterial (E. coli) lawn containing worms.
Worms eat bacteria engineered with DNA plasmids (feeding strains).
Plasmids are taken up by the worm’s intestinal cells.
Plasmids trigger destruction of the specifically targeted mRNA in the
intestinal cells.
The effect spreads to other cells. The specifically targeted mRNA is
destroyed in the whole animal.
RNAi by Feeding
E. coli
Feeding Strain
Gene Silenced “Dumpy”
Phenotype
Conducting RNAi by Feeding
Students will:
• Demonstrate the power of silencing a single gene
• Learn about a method for determining gene function
• Be introduced to a model organism
• Engage in bioinformatics exercises
• Explore Nobel Prize-winning research
Inducing RNAi by Feeding Kit
Item #211391
Making the Connection: Applying RNAi
dsRNA
RNAi Therapeutics: ALN-TTR02
• Impacts ~50,000 people
• 5 to 15 years’ survival after diagnosis
• Phase I trial
- 94% knockdown
• Phase II trial
- 2013 completion
B.U. Medical Center, July 2012
Disease target:
TTR-mediated amyloidosis
RNAi Therapeutics: ALN-AT3
• Impacts ~50,000 people
• Non-human primate
- 80% knockdown
• Phase I trial
- Late 2013 start
Adapted from 54th ASH meeting poster, Alnylam Pharmaceuticals
Disease target:
Hemophilia
RNAi Therapeutics: Path Forward
• Enabling technologies
- Delivery mechanisms
• Market perception
- $2.5−$3B invested
- No commercial products
• Personalized medicine
C. elegans Lab Kits
Culturing and Observing C. elegans Kit Item #211390
Inducing RNAi by Feeding Kit
Item #211391
Examining the RNAi Mechanism Kit
Items #211392 to #211394
Intro Intermediate Advanced
Genome Science
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