To identify potential SOX10 target genes, we will analyze the effects of altered SOX10 expression on the expression of multiple genes simultaneously using cDNA expression microarrays. Successful microarray experiments require: 1) appropriate mRNA populations to compare, 2) appropriate cDNA populations on the array, 3) critical secondary screens, and 4) defined functional assays. 1) We will compare expression patterns from cell cultures with altered SOX10 function. These will include primary cultures of SOX10Dom/+ versus +/+ neural crest and NC-Ms as well as cell lines transduced with SOX10 expressing viruses. 2)We are developing a standard set of mouse cDNA clones to use in microarray expression analyses based on the WASHU-Merck mouse ESTs. However, we have found that these generalized arrays may not be the most efficient source of cDNA for a particular tissue source. Therefore, we developed a novel strategy to isolate a set of cDNA clones representing the majority of genes that regulate NC-M development and function (Loftus et al, 1999). We will use this set for expression profiling in collaboration with Dr. Trent and the NHGRI microarray core. 3) Since we are screening for target genes of SOX10 transcriptional regulation, we will assess candidates by in situ hybridization using mutant SOX10 embryos at the time when SOX10 expressing cells are present but failing to migrate. Genes exhibiting a NC expression pattern in WT but not in mutant SOX10 expressing cells will be analyzed further. 4) Most in vivo approaches for defining gene function (transgenic and knockout strategies) require extensive cloning, viable offspring, and animal breeding. We will use our NC infection strategy described above (RCAS) to misexpress candidate gene products in NC and examine the effects on development of specific lineages. We have already identified one gene that results in a 4-fold increase in melanocyte number when melanoblasts are specifically targeted. - biotechnology research, cancer research, digestive diseases, gene mapping(non-human), neuroscience, pediatric research
Showing the most recent 10 out of 28 publications
