RNA interference is a method of choice to inactivate genes in various systems, including mammalian cells in culture. Unbiased, systematic screening of the genome by RNA interference offers the possibility to identify genes essential for any given function that can be assayed in cultured cells.
The library: siRNA against individual genes are arrayed in 384 wells plate and the entire human genome is covered with ~70 of these plates.
A 384 wells assay plate, an essential tool for large-scale RNAi screening (and a nightmare for manual pipetting)
The robot: never tired of pipetting
RNAi-Global: a large collaborative effort to develop the RNAi screening technology
IMCB has joined a consortium of laboratories involved in large-scale RNAi screening, the RNAi Global Initiative. This collaborative effort fosters the development of scientific standards and know-how to address the specific challenges of performing these large-scale experiments and interpreting their results.
Lab projects:
1. Metazoan genes required for constitutive secretion
Using Schneider's S2 engineered to secrete the Horse-Radish Peroxidase (HRP) enzyme, we previously interrogated the whole Drosophila genome to identify the genes required for constitutive secretion. This screen revealed a number of novel essential secretion genes with no equivalent in yeast (Bard et al. Nature, 2006).
Legend: A whole genome RNAi screen for secretion. 22000 different RNAi reagents were tested, the majority showed limited to no effect on secretion (score=0) while ~1000 showed a reduction of secretion..
2. Regulation of Golgi apparatus organization
The organization of the mammalian Golgi apparatus is highly complex and its regulation still poorly understood at the molecular level. We have developed an automated image-based analysis assay to assess perturbations in the human Golgi apparatus structure.
Automated quantification of the Golgi morphological parameters
3. Regulation of mucin granules formation
Mucin secretion is an instance of regulated secretion. The goblet cells in the mucosal epithelium are filled with mucin granules and will release their content upon stimulation only. We focus on the genes responsible for the formation of mucin granules. Deregulated mucin secretion can lead to impaired respiratory function, for example in asthma and cystic fibrosis. A better knowledge of the mechanisms regulating the formation of mucin granules and mucin secretion could lead to the development of new treatments for these diseases.
A cluster of human goblet cells :
(In blue: DNA staining, in green, mucin5AC staining)
(C) Copyright 2008 Institute of Molecular and Cell Biology, A*STAR Singapore.
