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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