Over the past decades, a great deal has been learnt about many mitotic genes and how they function in certain aspects of mitosis. How all mitotic genes coordinate their actions to achieve mitosis in a highly regulated fashion is, however, poorly understood. As the first step towards a global understanding of mitosis at a molecular level, MitoCheck sets out to search for all genes required for mitosis in human cells by two means. First, we will extract information from the literature and other public databases. Second, we will carry out genome-wide screens to systematically search for mitotic genes. The objective of this workpackage is to generate the tools for the identification of mitotic genes in human cells.

1) to compile from the literature, public databases and knowledge generated within the consortium labs a list of genes that are known to be required for mitosis.

2) to generate a genome-wide library of interfering RNAs for screens in human cells to identify mitotic genes.

3) to establish methods for delivering interfering RNAs into human cells.

Richard Durbin (Wellcome Trust Sanger Institute, Hinxton, UK)

Tony Hyman (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany)

Jan Ellenberg (EMBL, Heidelberg, Germany)

1) Picking of known genes:

The Sanger team has generated a web-accessible and fully searchable database that contains all known human genes, information about their corresponding transcripts and siRNAs, links to other public databases and manually-curated information from the literature. In the future, this database will also include movies from the genome-wide RNAi screen as well as information about crucial mitotic protein complexes and their cell cycle dependent phosphorylation state.

2) Production of a genome-wide library of interfering RNAs in human cells

We carried out systematic comparison of three alternative methods for producing interfering RNAs: chemically synthesized small interfering RNAs (siRNAs), short hair-pin RNAs (shRNAs) expressed from plasmids and endoribonuclease-prepared short interfering RNAs (esiRNAs). A genome-wide siRNA library was chosen for the primary screens in human cells. However, we continue to develop the esiRNA technology as it provides a renewable RNAi resource available to the academic community in the future.

3) High-throughput delivery of RNAs into human cells

We compared automated liquid phase transfection in 96-well plates with solid phase transfection using cell arrays. We decided to use the latter as the means for high throughput delivery of siRNAs into human cells in the genome-wide screen, mostly because it uses much less RNA per transfection, shows no detectable cytotoxicity and allows much higher throughput in microscopy based phenotyping.

- An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.
Kittler R, Putz G, Pelletier L, Poser I, Heninger AK, Drechsel D, Fischer S, Konstantinova I, Habermann B, Grabner H, Yaspo ML, Himmelbauer H, Korn B, Neugebauer K, Pisabarro MT, Buchholz F. Nature. 2004 Dec 23;432(7020):1036-40 (PMID: 15616564)

- Functional genomic analysis of cell division by endoribonuclease-prepared siRNAs.
Kittler R, Buchholz F. Cell Cycle (2005) 4:564-567) (PMID: 15876870)

- Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells.
Kittler R, Heninger AK, Franke K, Habermann B, Buchholz F (2005) Nat Methods 2:779-84 (PMID: 16179925)