The European Molecular Biology Laboratory is approximately halfway through a human genome-wide siRNA screen to identify genes involved in cell division, the research organization said this week.
The screen, which is expected to be completed by the end of the year, is part of a larger project called MitoCheck that is focused on determining how phosphorylation regulates mitosis in human cells.
"We're using a combined approach of functional genomics, chemical genomics, structural biology, and, in the end, pharmacology" to investigate mitosis, Jan Ellenberg, group leader at EMBL and a MitoCheck co-initiator, told RNAi News this week.
The first step of MitoCheck, he said, is a genome-wide RNAi screen "to identify genes that are necessary for cells to divide. Those are obviously the interesting ones to look at [to see if] the proteins [they make] are regulated by phosphorylation in their activity," he said. "There are many genes that are phosphorylated, but if they don't make any difference for cell division, then they are interesting in the context of mitosis."
"The phenotypic data saying a gene is or is not important for mitosis will be directly visible in Ensembl. You go in the genome database, you click on your favorite human gene, and you can directly see the results of our screen."
Supplying the siRNA library for this screen is Ambion, which was chosen over "several companies" by MitoCheck, Ellenberg said. He noted that a "decisive factor" in the selection of Ambion was the oligo design algorithm the company uses, which was created by EMBL spin-out Cenix BioScience.
"I know the people [at Cenix] very well, and I know they do a good job with the bioinformatics [for siRNA] design," he said.
Terms of the deal with Ambion were not disclosed.
MitoCheck was established in 2004 and is a four-year effort being funded by a roughly €8.5 million ($10.3 million) endowment from the European Union. Its members include the Research Institute of Molecular Pathology in Austria; EMBL; Deutsches Krebsforschungszentrum in Germany; Leica Microsystems Heidelberg; the Max Planck Institute of Molecular Cell Biology and Genetics; Gene Bridges, based in Germany ; the European Institute of Oncology in Italy; Centre de Recherches de Biochimie Macromoléculaire in France; Cancer Research UK; the University College London; and the Wellcome Trust Sanger Institute in the UK.
"We invested a year in technology development where we set up the infrastructure to do live-cell, microscopy-based, genome-wide screens — something that is not routinely done," Ellenberg said. "Now, that is fully underway. We're in the middle of the genome-wide screen at the moment."
He noted that, "conservatively, [the screen] should be done by the end of this year, [but] I hope earlier [since] our part of identifying the genes … is a prerequisite for other aspects of the project that start afterwards."
Ellenberg said that once the screen is completed, "we will then work on the function of the genes we identified, which we expect to be not more than roughly 1,000 genes."
Even though the RNAi screen will wrap up long before the overall MitoCheck project is concluded, the gene-silencing technology has additional roles to play in the effort.
"We will basically do two more things with RNAi," Ellenberg noted. "One is, once we have identified the gene [involved in cell division], we'll do secondary screens [using RNAi] where we not only knock out the genes specifically and individually, but do more complicated phenotypic assays.
"We will look in much more detail at mitosis with live-cell microscopy for the genes we identified … by knocking them out one by one and looking at many different fluorescent reporters we have for cell division," he said. "We will get quite a bit of cell biological information on these genes that were identified during the first screen."
Ellenberg said that assays for the secondary screens are under development, and that this work is slated to begin at the beginning of next year "when we have a reasonable number of candidates from the primary screen."
The other area where RNAi will come into play will involve a subset of genes selected as being the most relevant for cell division and mitosis. Cells "will be tagged with GFP and affinity tags," Ellenberg said. "Then the endogenous gene, which is not tagged in these cells, will be removed by RNAi. Then these cells will be looked at by a biochemical analysis [and] imaging analysis."
He said that the technology for this step has already been developed by the Max Planck Institute, which will handle the analyses, "and they are basically now waiting for us to complete the genome-wide screen [in order to] deliver to them the key candidates that are new and have striking phenotypes."
Overall, MitoCheck "is something like a pipeline where one project feeds the other to do subsequent analysis," Ellenberg said.
Data from MitoCheck "will be in a publicly available database, and also through [the MitoCheck] webpage" at www.mitocheck.org, Ellenberg said, adding that "at the moment, there's not much on [the site] because we haven't yet published the screen."
Additionally, genome annotation data will be made available through the Ensembl genome database. Richard Durbin, the MitoCheck leader at the Wellcome Trust Sanger Institute, is a co-creator of the Ensembl database, Ellenberg noted.
"The phenotypic data saying a gene is or is not important for mitosis will be directly visible in Ensembl," he said. "You go in the genome database, you click on your favorite human gene, and you can directly see the results of our screen."
— Doug Macron ([email protected])