Methodologies and resource generation enabling high-throughput, large-scale recombineering for high-fidelity gene expression analysis in C. elegans. (360G-Wellcome-082603_B_07_Z)

Reporter fusions are used extensively to determine C. elegans gene expression patterns. The applicants have advanced this technology by establishing a straightforward recombineering technique for seamless reporter insertion into fosmid clones. This advance allows trivial manipulation of large genetic units, with minimal disruption of the genetic material such that reliable data can be generated. We plan to: further develop our recombineering technique to increase throughput by evolving i t to 96-well pates initially with manual processing and subsequently using robotics characterize expression patterns of C. elegans genes within operons to reveal the complexities of this significant mode of C. elegans gene organization examine the distribution and function of the alternative gene products for C. elegans forkhead transcription factor genes to unravel the intricacies of these key control genes determine the expression patterns of all C. elegans winged helix and bHL H transcription factor genes as an important contribution towards construction of a model of the regulatory network controlling expression of the C. elegans genome provide these reagents to the wider worm community In this way we will further develop our recombineering protocol to answer important questions concerning the co-ordination of C. elegans gene expression.