Unit: DNA Micro-arrays (Platform)
Director: Coppee, Jean-Yves
The aim of the platform is to develop biochips technology for the study (comparative genomics and gene expression profiling) of model microorganisms and infectious diseases (pathogen microorganisms and response of the host to the infection).
We have the equipment for making and using DNA chips on various supports (glass slides, nylon membranes, Affymetrix chips). We are also developing the bioinformatical and statistical tools necessary for the analysis, interpretation and handling of the results.
The availability of the complete genome sequences of a growing number of microorganisms and the emergence of new technologies for the study of gene expression lead the Pasteur Institute to set up a platform dedicated to the fabrication and analysis of biochips for the study of model microorganisms and infectious diseases. These studies involve both the agents responsible for these pathologies (comparative genomics and gene expression profiling) and the response of the host (gene expression profiling).
At present, three technologies are used at the platform :
Microarrays : spotting of PCR products or long oligos onto glass slides and hybridization with fluorescent probes
Macroarrays : spotting of PCR products onto nylon membranes and hybridization with radioactive probes
Affymetrix : use of commercial chips containing in situ synthesized short oligos
The platform is in charge of projects selected by internal calls for proposals. The projects are made as collaborations. Most of the selected projects have national or european frameworks. The costs of fabrication and use of the chips are supported (partially or totally) by the Genopole.
For the macroarrays and the microarrays, the platform is in charge of:
- the preparation of the chips: design of the oligos (primers for PCR and long oligonucleotides), PCR amplifications, spotting onto solid supports, quality control of the spotted arrays
- the training of the users for the preparation of the labeled targets (fluorescent or radioactive), the hybridizations, the detection and the quantification of the hybridization signals.
For the projects based on the Affymetrix technology, the platform is in charge of:
- the preparation of the labeled targets (quality control of the nucleic acids and labeling)
- the detection and quantification of the hybridization signals
The analysis and the handling of the results is always performed in close collaboration with the users. Several tools allowing for the normalization of the data and their statistical analysis are installed at the platform. We are essentially working with the R environment (Bioconductor) and with several commercial softwares (Arrayminer, S-Plus-ArrayAnalyzer...).
In collaboration with S. Moreira and I. Moszer (Plate-forme Integration et Analyse Genomique), a database has been built according to the MIAME recommendations for the storage of microarrays and macroarrays data.
Some of our equipments are freely accessible to the campus : these include systems for checking the quality of the nucleic acids (BioAnalyzer, Agilent), of the labeled targets (Nanodrop, spectrophotometer for microplates) and RT-PCR device (Applied Biosystems 7900).
Projects centered on pathogens and model microorganisms:
In 2005, the platform has been involved in several projects aiming at the gene expression profiling or comparative genomics of pathogens.These projects are based on the macroarrays and microarrays technology. Each project is realised by a member of the platform in close collaboration with the requesting team. They have been selected following calls for proposals. Most of the projects last for more than one year. The complete list of all these projects is available on the website of the platform.
These projects concern in particular the complete genomes of Bacillus anthracis (coll. M. Mock), Bordetella (coll. N. Guiso), Candida glabrata (coll. B. Dujon), Entamoeba histolytica (coll. N. Guillen), Legionella pneumophila (coll. C. Buchrieser), Plasmodium falciparum (coll. P. David), Staphylococcus aureus (coll. T. Msadek), Streptomyces lividans (coll. P. Mazodier) et Yersinia (coll. E. Carniel).
These projects aim at answering the following biological questions:
- study of virulence factors
- inter-species transcriptomes comparison
- study of the host-pathogen interactions
- study of the effects of environmental pressures (stress, immune pressure, drug pressure...) on the pathogen
- identification of regulators targets (virulence, motility...)
Projects centered on higher eukaryotes:
The majority of the projects dealing with higher eukaryotes are based on the Affymetrix technology. This allows the in situ synthesis onto a silicium support of short oligonucleotides by a photolithographic process. These are high density chips. For example, on the latest generation chips, 54 000 probes interrogate 47 000 transcripts for the whole human genome.This technology allows for gene expression profiling, molecular genotyping and polymorphism detection. Several projects have been selected following calls for proposals and, as for the microarrays technology, most of them last for more than one year. The selected projects receive fundings (2/3 of the cost of the project being supported by the Pasteur Institute). The biological questions studied with this technology are centered on the host response to the infection and on biology of the development. 13 projects have been selected in 2005. The complete list of the projects is available on the website of the platform.
The platform has also been involved in two other projects dealing with higher eukaryotes and based on the microarray technology. These projects aim at the development of DNA chips for the microRNAs expression profiling in Drosophila (coll. C. Antoniewski) and of genomic chips for the study of the murine X chromosome inactivation center (coll. P. Avner).
The platform participates to several Pasteur Courses (Molecular and Cellular Genetics, General Microbiology, Mouse Molecular Genetics, Proteins Biochemistry). We have also organized permanent trainings (at the Pasteur Institute and at the Louis Pasteur University in Strasbourg). The platform has also organized a one-week training dedicated to the analysis and handling of DNA chips data.
Keywords: Transcriptomics, comparative genomics, infectious diseases, macroarrays, microarrays, Affymetrix