2. Introduction to C. elegans
and
RNA interference

3. Why study model organisms?

4. 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

5. How do geneticists study gene function?

6. Disrupt the gene and analyze the resulting phenotype
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

7. Why study mutants in model organisms?

8. 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

9. Sort through the mutations identified
Linkage mapping and complementation analysis.

10. 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

11. What makes a good model organism?
Ease of cultivation
Rapid reproduction
Small size

12. Caenorhabditis elegans
The model organism:
Caenorhabditis elegans
Electron micrograph of a C. elegans hermaphrodite

13. 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

14. C. elegans cell division can be studied in the transparent egg

15. C. elegans cell lineage is known

16. Nuclei and DNA can be visualized
Kelly, W. G. et al. Development 2002;129:

17. 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

18. 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)

19. Disrupt the gene and analyze the resulting phenotype
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

20. Let’s see how similar our genes are
to model organisms

21. Many genes are conserved in model
organisms
Species
  Number of Genes  
HomoloGene
Input
Grouped
groups
                                                                                                                                                                         
H.sapiens
23,516*
19,336
18,480
P.troglodytes
21,526 
13,009
12,949
C.familiaris
19,766 
16,761
16,324
M.musculus
31,503 
21,364
19,421
R.norvegicus
22,694 
18,707
17,307
G.gallus
18,029 
12,226
11,400
D.melanogaster
14,017 
8,093
7,888
A.gambiae
13,909 
8,417
7,882
C.elegans
20,063*
5,137
4,909
S.pombe
5,043 
3,210
3,174
S.cerevisiae
5,863 
4,733
4,583
K.lactis
5,335 
4,454
4,422
E.gossypii
4,726 
3,944
3,935
M.grisea
11,109 
6,290
5,884
N.crassa
10,079 
5,908
5,902
A.thaliana
26,659 
11,180
10,857
O.sativa
33,553 
11,022
9,446
P.falciparum
5,222 
971
950

22. Can the function of a gene be studied when all we have is the DNA sequence?

23. 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)

24. 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

25. 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

26. With the completion of the genome sequencing project, a quicker, less expensive reverse genetics method was needed. Luckily scientists discovered . . .
RNAi

27. RNAi

28. How did we come to understand how RNAi works?
Examining the antisense RNA technique revealed that the model for how it worked was wrong.

29. The old model: Antisense RNA leads to translational inhibition
mRNA is considered the sense strand
antisense RNA is complementary to the sense strand

30. The old model: Antisense RNA leads to translational inhibition
This can give the same phenotype as a mutant

31. 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

32. 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".

33. 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.

34. 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)