Participants involved: Berlin (responsible), Paris.
Collaborating institutes: Amsterdam.

Objectives

The goal of this workpackage is to dentify novel regulatory pathways and gene cascades that play a role in the pathogenesis of MRX.

Methodology and study materials

Employing state-of-the-art arraying and confocal laser scanning equipment as well as data analysis software, we will expand and use already established cDNA arrays, but also generate additional ones. A murine adult brain array with more than 3,000 clones will be expanded, and we will generate a murine fetal brain array containing more than 10,000 brain-expressed genes. The clones will be spotted in triplicate for normalisation of the signals (participant Berlin).

Generation of fluorescent (Cy3/Cy5) probes derived from total RNA from the brains of MRX animal models and control animals (work package 5). In addition, probes will be generated from primary neuronal cells transfected with MRX-gene expression vectors and empty vectors (control) will be hybridized to the cDNA microarrays. Dye-swapping experiments will be performed to exclude false-positive clones (all participants of this work package).

Except for the "10,000 murine" array, the clones on our cDNA arrays are only partially sequence-verified. Therefore, it is still necessary to confirm the identity of all differentially expressed clones by sequencing (participant Berlin).

Gene expression profiles will be determined for the investigated neuronal cells and animal brains. For the latter, brain regions that show high expression for the relevant MRX genes will be selected. Preliminary studies have revealed high expression of OPHN1 and PAK3 in perception corticies, hippocampus and olfactory bulb. For oligophrenin, a high level of expression is observed in the cerebellum and expression profiling will also be performed using RNA prepared from the cerebellum. The comparative analysis will not only reveal individual genes but also entire genetic cascades, which will be key to understanding the pathogenetic mechanisms that give rise to MRX. Thus, these studies will provide important new insights into the function of the normal and aberrant brain and may have implications for the treatment of X-linked and other forms of mental retardation (participant Berlin).