Download Functional genomic analysis of cell division in C. elegans

High throughput genetics &
RNAi Screens
Luke Lopas
and
Mark Devries
RNAi history
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/RNAi-Epigenetics-and-Gene-Regulation/RNAi/RNA-Interference-Overview.html
Mechanisms of RNAi
http://www.nature.com/nrg/journal/v5/n8/pdf/nrg1415.pdf
C. elegans
Back ground
• Development to adult in about 2.5 days
• Life span 2-3 weeks
• Either hermaphroditic or male
• Why is it such a good models organism?
. Grows fast
. Shares similar cellular components
. Transparent
. All 959 somatic cells traced
Different methods of RNAi
Microinjection
• Most potent method
• Difficult method
Soaking
• High throughput analysis
• Can conduct stage specific analysis
• Weaker effect
Feeding
• High throughput analysis
• Weak effect
Transgene
• Allows for inducible/ tissue specificity
• More effective method for some genes
Choosing a gene library
•
•
•
•
•
•
•
Genomic sequence information
Predict wrong genes structure
Only 9% of genome not covered
cDNA
Can have stronger effect
Only represents genes being expressed
Misses low expression genes
Functional genomic
• Aims to give in vivo function to every
gene
• Uses all techniques of RNAi delivery
• Uses both types of RNAi libraries
• Requires a dissecting microscope
• Can use rrf-3 mutant
Functional genomic findings
• Insight into genome organization in C.
elegans
• Highly expressed genes show
phenotype
• Chromosomal regions have specialized
function
Short comings
• Protocol difference
• Inter experimental variability
RNAi gene screen
. Focus on specific process
. Reverse genetics vs forward genetics
Genome
wide
. whole
genome
Gene family
. Specific
gene family
. Narrows
choices
Transcriptome
.Expressed
genes
.Microarrays
Interactome
. proteinprotein
. Yeast two
hybrid
Use
functional
genomics
. Narrows
choices
. ‘in Silico’
Functional genomic analysis
of cell division in C. elegans
using RNAi of genes on
chromosome III
Gonczy et al.
What did they want to do?
• “…apply RNAi on a genomic scale to identify
genes required for cell division.”
– Unbiased testing of each ORF…actually testing
96% of ORFs
• Use a functional genomic approach in early
life cycle of C. elegans to identify genes
necessary for cell-division
– Specifically investigate genes on chromosome III
• Assign cellular functions to the multitude of
genes coming out of sequencing projects.
Why RNAi?
• Easy experiment
(relative)
• Can study cell
division
– No mitosis occurs
between dsRNA
innoculation and
fertilization
Picture from Ahringer Lab
How’d they do it?
Figure From Ben Schmidt’s 875 presentation
How’d they do it? Continued
Figure from Ben Schmidt’s 875 presentation
Data analysis
DIC microscopy used
for first 24 hours
- requires transparent
samples
Effects on progeny
observed over 2-4
days
Micrasterias radiata as imaged by DIC microscopy, from Wikipedia
Controls
From Gonczy et al., 2000
Results
From Gonczy et al., 2000
More Results
From Gonczy et al., 2000
How does this tie into what
we’ve done before?
Imagine the categories on the previous
slide…do they remind you of anything?
How does this tie into what
we’ve done before?
Imagine the categories on the previous
slide…do they remind you of anything?
Gene Ontology Categories
Analysis of Chromosome III
• Tested 2,232 ORFs
(96.4%)
– 133 (6.1%) gave
detectable DIC
phenotypes
• Found all previously
known mutations
from classical
genetic studies
University of Edinburgh
Analysis of Chromosome III
• RNAi loses potency
later in development
• In total: tested 12%
of ORFs in genome,
so if assume
random genomewide distribution,
over 1,000 genes
essential for first two
cleavage divisions!!!
University of Edinburgh
Is RNAi an efficient tool for gene
function discovery?
Pros
- Can look at a ton of
genes
- Can ascribe function to
genes w/o previously
ascribed function
- Provides a source for
investigation using
classical genetic
methods
Cons
- Loss of sensitivity as
development
proceeds
- Questions
surrounding
neuronal sensitivity
DIC images
From Gonczy et al., 2000
Guess the phenotype
• First video is wt
• All other videos
have a
developmental
phenotype.
C. elegans movies
Answers
All are mutants in different alleles in the
same gene. Defect involves cell
division, more specifically spindle
formation.
Can these results be
translated to other organisms?
The gene implicated in
cell division are
more highly
conserved among
species
From Gonczy et al., 2000
What do results mean?
From Nature Networks
Take home messages
• Lots of genes are involved in early
development!
• RNAi allows you to look at and assign
putative function to A LOT of genes!
• RNAi is an efficient reverse genetic tool
but is not perfect
Silencing Genomes
Phenobank
Phenobank
Phenobank
Phenobank
Movie Link
Other interesting websites
• Drosophila RNAi Screening Center
– If you are at all interested in fly RNAi and
how these results translate to mammals,
check it out!
Question from Class
•
•
I have two general questions with regards to functional genomic
screens and RNAi; first of all, we learnt from gene ontology that it
encompasses three distinct processes - is RNAi used as a method to
derive molecular function and/or biological processes information for
gene ontology?
Secondly, RNAi requires a gene to be knockout to identify the function
of that particular gene; what about genes that have redundancy
function, i.e. more than one gene performs the same function? The
possibility of redundancy function or synergistic function (where
function can be measured along a scale - the more genes that code for
a function is present, the more gene phenotype is observed) seems to
me that it renders incomprehensive screens of gene function.
– This was one stated advantage of using C. elegans, the fact that there is
little redundancy in genome. But what do you think about studies in
mammals?
Question from class
•
ITs obvious that RNAi is becoming a powerful tool in worm research,
but what are the implications to humans? I've read some papers on
RNAi therapeutics, and was curious about your thoughts on that?
Could ones' genome be sequenced, bad things identified, and then
specific RNAs given to silence problematic genes? This is almost like
gene therapy which could someday become more of a reality.