Work Package 4

Development of rapid assays to score developmental mutants

Introduction
In collaboration with Minos Biosystems UK, we have developed and are presently optimizing a fly transposition system in the mouse to rapidly generate mutants. In each of these mutants the mutation is marked by a transposon allowing rapid identification of the affected gene. Using lentiviruses, 50-100 ES cell lines (and mice derived from the ES cells) carrying different positions of lentivirus integrations equally spread across the mouse genome are created. This set serves as a starter set of ES cells or mice to start a so-called “portable” library of ES cells and mice by crossing males undergoing sperm transposition. We propose to develop robust but simple methodology that allows rapid analysis of large numbers of mutant mouse embryos in a library made with the transposon that contains an exon trap (to interrupt genes and express a fluorescent marker GFP) and a tet-inducible antisense promoter to inhibit the second allele of the interrupted gene. Breeding a large number of mice would result in covering most or all of the genes in the mouse for one of these types of mutations.

Objective
The development of methodology to rapidly identify large numbers of (mutant) embryos that have a defect in a developmentally important gene and have these available to all of the participants. Embryos screened at one of the locations will electronically be available to all others. The data collection will take place and to share these data within the network.

Approach
The project would be started by scoring developmental mutants by classical phenotypic or physiological abnormalities to establish proof methodology. At the same time we would start devising a set of criteria using known mutants that would allow scoring of the embryos for all developmental mutants of interest to this network. We would also work, in collaboration with the European Imaging Network applying under FP6, to screen embryos by 3D tomography (eg. James Sharpe, Edinburgh) and 3D GFP expression (FORTH) and develop a communication network to allow all partners instant electronic access to the 3D embryos that have been analysed.

Links with other projects: To almost all other projects in Themes 1, 2 and 3. Keywords: developmental mutants, 3D imaging

Participants: Grosveld, Minos Biosystems UK, European Imaging Network KP6