Dear
All,
We are happy to announce that Despina Siolas will
be defending her dissertation on Tuesday, July 29, 2008 at 2:00 P.M.
at the Bush Auditorium, Cold Spring Harbor Laboratory
ADVISOR: Dr. Greg Hannon
Cold Spring Harbor Laboratory
The Graduate Program in Genetics
Abstract:
Title:
"RNA interference Screens as a Tool for Discovering Gene
Function"
Recent advances in the field of RNA
interference (RNAi) have enabled researchers to conduct in depth investigations
of gene function. High throughput screens in cultured mammalian cells
can now be performed using RNAi libraries such as our own Hannon-Elledge
library. Our second-generation shRNA libraries consist of over 200,000
short hairpin RNA constructs targeting over 45,000 human and mouse
genes modeled after primary miRNA transcripts. Each hairpin is linked
to a unique 60 nucleotide identification sequence, which serves as
a barcode and allows us to virtually count the number of cells that
contain a specific hairpin in a cell population. Small changes in
barcode copy number can be monitored through the use of microarray
technology. The barcode can be amplified from a cell’s genomic
DNA and fluorescently labeled to produce a probe that is hybridized
to a microarray. We have optimized our probe labeling methods, probe
size and hybridization conditions using library plasmid DNA in a Nimblegen
platform. This optimized protocol allows us to detect 78.6% of probes
within 1 standard deviation above the mean background from a complex
mixture of approximately 1500 hairpins. In addition, by using two
color hybridization (Cy3 and Cy5) we can detect control subsets of
hairpins known to be depleted from a sample population. I applied
this RNAi barcode screening method in a screen using a complex mixture
of 7500 library hairpins in a p53 isogenic HCT 116 colon cancer cells
to identify genes that modify sensitivity to a common chemotherapeutic,
doxorubicin. Knockdown of Chk1, a kinase that mediates the G2/M checkpoint,
increases sensitivity to doxorubicin in the p53 deficient HCT116 cells
but not to the p53 wt HCT116 cell line. This work illustrates the
powerful use of RNAi screens to search for genes that synergize with
existing therapeutics, and contributes to genetically informed combination
therapies.
