Download blumberg-lab.bio.uci.edu

Timothy Abreo and Mrinal Sinha
Goal:
Ok, so we have the whole genome sequence of
Drosophila Melanogaster and we have mapped
its genes--now what?
benchfly.com
http://ocasapiens-dweb.blogautore.repubblica.it/
Model Organism--Drosophila Melanogaster (DM)
We are going to look at very specific cell
culture--particularly DM hemocytes
Specific Mechanics
RNAi = RNA Interference
Specifically with dsRNA--now known as small
interfering RNA (siRNA).
quickmeme.com
How does RNA interference work?
Genequantification.de
A specific example:
Gene called D-IAP1.
It is an anti-apoptosis gene.
How D-IAP1 Became the Positive Control
Functional Assays
● Flow Cytometry
● Sytox/Hoesht staining
Ok, so what?
galleryhip.com
Well...
We know what D-IAP1 does, and when we took
it out, we saw our expected phenotype.
But...
We are going to look at very specific genes,
specifically ones related to cellular growth and
survival.
scitechdaily.com
Then we add this to a bunch of cells-
Interpro Protein Domain Analysis
Company’s Description:
InterPro provides functional analysis of proteins by
classifying them into families and predicting domains and
important sites. We combine protein signatures from a
number of member databases into a single searchable
resource, capitalising on their individual strengths to
produce a powerful integrated database and diagnostic
tool.
Phenotypic Screens
● 13 % Ribosomal genes and 8% Proteosome genes found
● Cell cycle genes only found significant in S2-R cells
● Most populated section with 14% were genes encoding
proteins with DNA binding domains
● DNA binding domains included chromatin related factors
and transcription factors
Analysis of Essential Genes Functions
● Focused on 438 transcripts with z scores >3
○ only 20% of these genes had associated mutant alleles
○ 47% had associated gene ontologies
○ Interpro: 59% identified protein domains, 37% in severe cases
Analysis of Two Specific LOF Genes Identified
● Two genes found with similar z scores to
D-IAP1
○ CG11700: ubiquitin-like gene
○ CG15455: encodes acute myeloid
leukemia-like transcription factor
● Sytox and Hoesht analysis revealed 95
% of cells treated with dsRNA for
CG11700 or D-IAP1 were apoptotic
● 20% of cells apoptotic when treated with
dsRNA for CG15455
● Flow cytometry revealed decreased cell
size and DNA content, indicative of
dying cells
Rescue of the Two LOF Phenotypes
● Cells treated with an inhibitor of proapoptotic caspase (Nedd2-like)
reverted the cell death in response to RNAi for either CG11700, DIAP1, or (to a lesser extent) CG15455
● Same phenomena was observed when cells were treated with
RNAi targeted at proapoptotic caspases, except in cells with coRNAi for CG15455
● Demonstrated to researchers that CG11700 and D-IAP1 were likely
involved in the same pathway
Ortholog and Homolog Search
● Percentage of predicted orthologs with RNAi analysis was higher
than those found by database searches
● 50 genes had homology to human disease genes, with 10
associated with blood-cell leukemia
o Demonstrated that functional RNAi analysis in Drosophila can
discover common genes involved in cell viability of Animals
● However, in severe RNAi phenotypes, few homologs were found
identified amongst yeast and animals
o Suggests that metazoans evolved specific mechanisms
involved in cell viability
Implications and Future Applications
● Functional RNAi analysis can reveal unknown gene and
evolutionary conserved gene functions
● Quantitative nature of this analysis facilitates detection of gene
functions with subtle or redundant phenotypes via inducing a LOF
● Allows for statistical clustering in comparing several cellular
phenotypes to determine gene functions
● Genome wide RNAi can be utilized to screen multiple cellular
pathways and their various components, allowing for a better
understanding of systems involved in disease and development
Thanks...for staying awake!
Any Questions?