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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
Why study mutants in model organisms?
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.
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
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
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
The Genome Sequencing Project
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
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
Let’s see how similar our genes are
to model organisms
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
Can the function of a gene be studied
when all we have is the DNA sequence?
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
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
With the completion of the genome
sequencing project, a quicker, less
expensive reverse genetics method was
needed. Luckily scientists discovered . . .
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-stranded RNA 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
• Hypothesis: addition of
extra purple pigment genes
should produce darker
flowers.
• Results: Instead, the
flowers became whiter.
• New hypothesis: the
multiple transgene copies
of the pigment gene made
double stranded RNA.
C. elegans is amenable to many
forms of RNAi treament
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)