The objective of the proposed research is to identify and ascertain the function of novel genes involved in nervous system development. To accomplish this, I have developed an in vitro cell culture system that will allow me to efficiently identify, mutagenize, and clone genes that are expressed in the embryonic nervous system. Briefly, embryonic stem (ES) cells are transfected with a gene trap vector and induced with retinoic acid to differentiate into neurons in vitro. The gene trap randomly inserts into the coding region of genes, thereby disrupting their function and allowing for expression of a lacZ reporter in cells that express the disrupted gene. Differentiated neurons derived from transfected ES cells are screened for expression of lacZ, as an indicator that a given ES cell clone harbors a disrupted neuronal gene. CDNAs of trapped genes expressed in neurons will be generated with lacZ- specific primers, cloned, and subjected to rigorous molecular characterization. Preliminary results from this screen have yield two ES cell clones that each contain a disrupted allele for a novel gene, both of which are expressed in the embryonic and neonatal nervous system of the mouse. These cells will be injected into host blastocysts in order to make germline chimeras. The normal function of these genes may be inferred from morphological defects of transgenic embryos homozygous for the gene trap allele.
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| Lyons, G E; Swanson, B J; Haendel, M A et al. (2000) Gene trapping in embryonic stem cells in vitro to identify novel developmentally regulated genes in the mouse. Methods Mol Biol 136:297-307 |
