Introduction to C. elegans
and
RNA interference
Why study model organisms?
The problem:
• In order to understand biology, we need to
learn about the function of the underlying
genes
• How can we find out what genes do?
• We need a way to uncover these functions
How do geneticists study gene function?
How do geneticists study gene function?
Disrupt the gene and analyze
the resulting phenotype
Forward genetics:
• Classical approach
• A gene is identified by studying mutant
phenotype and mutant alleles
• The gene must be cloned for further
functional analysis
How do geneticists study gene function?
Disrupt the gene and analyze
the resulting phenotype
Reverse genetics:
• Start with gene sequence information
• Engineer a loss of function phenotype to
evaluate gene to function
Forward Genetics
Starting point: A mutant animal
End point:
Determine gene function
• Have a mutant phenotype and wish to determine what
gene sequence is associated with it
• Allows identification of many genes involved in a given
biological process
• Mutations in essential genes are difficult to find
• Works great in model organisms
What makes a good model organism?
Ease of cultivation
Rapid reproduction
Small size
Why are mutants in model organisms useful?
Let’s see how similar our genes are
to model organisms
A comparison of genomes
Model organism
Haploid
genome size
(Mb)
Estimated # of
genes
S. cerevisiae
13
6,022
C. elegans
100
14,000
A. thaliana
120 (estimated)
13,000-60,000
D. melanogaster
170
15,000
M. musculus
3,000
100,000
Homo sapien (not a
model)
3,000
100,000
Many genes are conserved in model
organisms
Species
H.sapiens
P.troglodytes
C.familiaris
M.musculus
R.norvegicus
G.gallus
D.melanogaster
A.gambiae
C.elegans
S.pombe
S.cerevisiae
K.lactis
E.gossypii
M.grisea
N.crassa
A.thaliana
O.sativa
P.falciparum
Number of Genes
HomoloGene
Input
Grouped
groups
23,516*
21,526
19,766
31,503
22,694
18,029
14,017
13,909
20,063*
5,043
5,863
5,335
4,726
11,109
10,079
26,659
33,553
5,222
19,336
13,009
16,761
21,364
18,707
12,226
8,093
8,417
5,137
3,210
4,733
4,454
3,944
6,290
5,908
11,180
11,022
971
18,480
12,949
16,324
19,421
17,307
11,400
7,888
7,882
4,909
3,174
4,583
4,422
3,935
5,884
5,902
10,857
9,446
950
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene
The model organism:
Caenorhabditis elegans
Electron micrograph of a C. elegans hermaphrodite
Caenorhabditis elegans
Profile
Soil nematode
Genome size: 100 Mb
Number of chromosomes: 6
Generation time: about 2 days
Female reproductive capacity: 250 to 1000 progeny
Special characteristics
Strains Can Be Frozen
Hermaphrodite
Known cell lineage pattern for all 959 somatic cells
Only 302 neurons
Transparent body
Can be characterized genetically
About 70% of Human Genes have related genes in C. elegans
C. elegans cell division can be
studied in the transparent egg
C. elegans cell lineage is known
Nuclei and DNA can be visualized
Kelly, W. G. et al. Development 2002;129:479-492
How do geneticists identify genes?
Answer: They perform a mutagenesis screen.
1. Mutagenize the organism to increase the likelihood of
finding mutants
2. Identify mutants
3. Map the mutation
4. Determine the molecular function of the gene product
5. Figure out how the gene product interacts with other gene
products in a pathway
Sort through the
mutations
identified
Linkage mapping and
complementation
analysis.
What are the limitations of Forward Genetics?
1. Some genes cannot be studied by finding
mutations
• Genes performing an essential function
• Genes with redundant functions
2. Finding mutants and mapping is time-consuming
3. Mutagenesis is random
• Cannot start with a known gene and make a
mutant
Genome sequencing has identified many
genes
Model organism
Haploid
genome size
(Mb)
Estimated # of
genes
S. cerevisiae
13
6,022
C. elegans
100
14,000
A. thaliana
120 (estimated)
13,000-60,000
D. melanogaster
170
15,000
M. musculus
3,000
100,000
Homo sapien (not a
model)
3,000
100,000
Can the function of a gene be studied
when all we have is the DNA sequence?
Reverse Genetics
Starting point: Gene sequence
End point:
Determine gene function
• Have a gene in hand (genome sequence, for example),
and want to know what it does.
• Can be used to correlate a predicted gene sequence to a
biological function
• Goal is to use the sequence information to disrupt the
function of the gene
Some approaches to Reverse Genetics
• Targeted deletion by homologous recombination
– Specific mutational changes can be made
– Time consuming and limited to certain organisms
• Mutagenesis and screening for deletions by PCR
– Likely to completely abolish gene function
– Time consuming and potentially expensive
• Antisense RNA
– Variable effects and mechanism not understood
A new, fast, generally applicable
technique was needed
And the winner is…..
RNAi
How did we come to
understand how RNAi works?
Examining the antisense RNA technique
revealed that the model for how it
worked was wrong.
The old model:
Antisense RNA leads to translational inhibition
mRNA is considered the sense strand
antisense RNA is complementary to the sense strand
The old model:
Antisense RNA leads to translational inhibition
This can give the same phenotype as a mutant
An experiment showed that the antisense
model didn’t make sense:
• The antisense technology was used in worms...
• Puzzling results were produced: both sense and antisense
RNA preparations were sufficient to cause interference.
• What could be going on?
1995
Guo S, and Kemphues KJ.
First noticed that sense RNA was as effective as
antisense RNA for suppressing gene expression in worm
When researchers looked closely, they found
that double-stranded RNA caused the
silencing!
Negative control
uninjected
Potent and specific
genetic interference by
double-strandedRNA in
Caenorhabditis elegans
Andrew Fire*, SiQun Xu*, Mary K. Montgomery*,
Steven A. Kostas*†, Samuel E. Driver‡ & Craig C. Mello‡
mex-3B antisense RNA
mex-3B dsRNA
Double-stranded RNA injection reduces the levels of mRNA
1998
Fire et al.
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".
dsRNA Hypothesis explains the
white petunias
•
Purple plants should become purpler...
•
Instead, they became whiter.
•
How could this be happening?
• The multiple inserted copies of
chalcone synthase were
producing double stranded
RNA
RNAi in worms: easier than baking pie!
We are going to inactivate genes by RNAi by feeding
•Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998).