The long-term objective towards which the current proposed work will contribute is the establishment of a random sequence tagged insertion library in C57/B6 embryonic stem cells from which cells carrying individual, known, integration events could be widely distribution to the scientific community.
Four specific aims are proposed towards this objective. First, we will refine a high throughput sequence tag acquisition technology, which we have invented, for use in an ES cell gene-trapping vector in an automated format. Second, we will generate an ES cell gene trap vector carrying the sequence elements required for the sequence tag acquisition technology and recombinogenic sequence elements (FRT sites) that will allow the sequence at a given integration site to be modified. As part of this aim, vectors that allow """"""""delivery"""""""" of alternative sequence elements to the integration site will be constructed and tested. Third, we will establish a repository of early passage C57BLI6JSvEvTac ES cell lines that retain the ability to contribute to the germ line in chimeric mice even after many passages. Additionally, methods for the automated isolation of ES cell clones will be tested. Finally, a demonstration library of 5,000 sequence tag indexed ES cell clones will be generated. The methodologies and reagents developed here will be fully sufficient to allow the rapid establishment of a random sequence tag insertion library of 100,000 or more events in which the location of each insertion sites is known.
| Pruitt, Steven C; Mielnicki, Lawrence M; Stewart, Carleton C (2004) Analysis of fluorescent protein expressing cells by flow cytometry. Methods Mol Biol 263:239-58 |
| Pruitt, Steven C; Bussman, Amy; Maslov, Alexander Y et al. (2004) Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro and in vivo. Gene Expr Patterns 4:671-85 |
