Page 170 - 53rd Annual Drosophila Research Conference

Full Abstracts – TECHNIQUES AND FUNCTIONAL GENOMICS
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Synthetic Genetic Interactions of Cell Cycle Modulators in Drosophila. Maximilian J. Billmann1, Thomas Horn 1, Bernd Fischer2 , Thomas Sandmann1 ,
Wolfgang Huber2 , Michael Boutros1 . 1) German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics, Heidelberg, Germany; 2)
EMBL, Genome Biology Program, Heidelberg, Germany.
To identify novel modulators of the cell cycle and understand their interactions, we performed a sensitive multi-parametric analysis of mitotic phenotypes
on a single cell level. To this end, we depleted almost every gene in the Drosophila genome and imaged Drosophila cells stained for Tubulin, DNA and
phospho-Histone 3 levels by automated fluorescent microscopy. Multi-parametric image analysis identified the vast majority of previously described cell
cycle regulators and pinpointed potential novel regulators displaying strongly aberrant mitotic ratio or nuclear size phenotypes. A time-course analysis
provided complementary insights into the dynamic manifestation of phenotypes. Novel candidates were validated for their in vivo role during Drosophila
wing development and intestinal stem cell maintenance. To derive specific predictions of candidate gene function, we conducted synthetic genetic
interaction experiments by combinatorial RNAi with putative novel and well-known cell cycle regulators. We found e.g. the uncharacterized cell cycle
modulator l(2)NC136 to cluster with known regulators of mitosis.
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The Transgenic RNAi Project at Harvard Medical School, The TRiP, is expanding the collection and is establishing the "Digital Red Book of
RNAi". LA Perkins1,2, L Holderbaum1, D Yang-Zhou1, L Jiang1 , R Tao1 , C Hu1 , R Sopko1 , S Ball1, M Foos1, A Miller1 , S Randklev1, I Flockhart 1, B
McElvany1 , S Mohr1 , JQ Ni1,4 , LP Liu1,4, S Kondo 1,5, N Perrimon1,3 . 1) Dept Genetics, Harvard Medical School, Boston, MA; 2) MGH, Boston, MA; 3)
HHMI; 4) Tsinghua U Stock Center, China; 5) DGRC, Japan.
In Drosophila expression of RNAi constructs using the Gal4/UAS system has emerged as the method of choice to determine the functions of all genes. To
facilitate in vivo RNAi studies, a number of large-scale resources have been developed that rely on long doublestranded RNAs (dsRNAs). These dsRNAs are
problematic as they do not work in the germline and many have poor knockdown efficiencies. The goals of the TRiP (http://www.flyrnai.org), funded by
NIH/NIGMS, were to improve methods of transgenic RNAi and to generate RNAi lines for the community. During our first funding period we have
optimized vectors for transgenic RNAi and shown that small hairpins (shRNAs) are better reagents than dsRNAs for in vivo RNAi, they're more effective in
somatic tissues and work in both germlines. Based on the growing need for shRNA fly lines, we have generated >6,000 fly stocks that are distributed by the
BDSC. During our second funding period, we will continue expanding the collection of TRiP shRNA lines, by generating lines ourselves (using GSI for
injections) as well as coordinating the production of lines by a number of outside groups (DGRC, Japan; Tsinghua U, China; and individual labs) that are
interested in helping build the resource. In addition, as information on the efficacy of RNAi lines is currently not being tracked, we are establishing
the “Digital Red Book of RNAi” by collecting information on existing lines and performing validation experiments (qPCR) to evaluate the performance of
existing and new lines. Well-organized public availability of this information will not only help individual researchers select the best lines for experiments
but also help us identify additional shRNA lines that need to be produced.
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Building a community resource of GFP tagged Drosophila melanogaster transcription factors. Rebecca F. Spokony1 , Alec Victorsen1 , Stacy L.
Holtzman 2, Sarah El Mouatassim Bih1 , Rebecca Cholst1, Nader Jameel1, Koen J.T. Venken3 , Michael Z. Ludwig1 , Jennifer Moran1, Nicolas Negre1 , Matthew
Slattery1, Hugo J. Bellen3 , Thomas C. Kaufman2 , Kevin P. White1 . 1) Institute for Genomics & Systems Biology, University of Chicago, Chicago, IL; 2)
Department of Biology, Indiana University, Bloomington, IN; 3) HHMI, Baylor College of Medicine, Houston, TX.
Using recombineering of BAC genomic fragments, we developed a set of transgenic Drosophila melanogaster lines expressing EGFP tagged transcription
factors (TTFs) regulated by their endogenous cis-genomic regions. These lines enable the study of proteins that would have previously been difficult due to a
lack of or limited reagents. The lines can be used to study a wide variety of biological questions including expression, regulation and protein-DNA binding.
We replaced the endogenous stop codon with EGFP within large fragments of genomic DNA (~30-100 kb) and integrated these into the genome at well-
characterized locations containing PhiC31 attP sites. We currently have 60 lines for 45 different transcription factors plus alternatively-spliced DNA-binding
isoforms and different genomic contexts. We are validating expression of the TTFs by comparing EGFP expression with published RNA and protein
expression for the various transcription factors . We are determining TTF function for a subset of the lines with mutant rescue. Almost all of the genomic
insertions can be maintained as homozygotes and a small portion of them show mild overexpression phenotypes. For example, Notch -EGFP adults display
the well-characterized Notch overexpression ectopic wing vein phenotype in a dose and temperature dependent fashion. Additionally, we are discovering
new expression patterns for genes with little or no published expression data that are consistent with known mutant phenotypes as well as new
overexpression phenotypes that are consistent with known expression patterns. We are currently using the validated lines for regulatory element dissection
(e.g., Jra) and ChIP-seq (e.g., Hox genes and nuclear receptors).