It is important that an Expressed Sequence Tag (EST) database produced for a model organism represents rnRNA transcripts from all possible tissues. We propose to use a transgenic approach to help identify cell lineage-specific ESTs from developing zebrafish embryos. The focus of this proposal will be the identification of ESTs from embryonic neuronal cells. To this end, we have generated transgenic zebrafish that express the green fluorescent protein (GFP) reporter gene specifically in embryonic neuronal cells. GFP-positive neuronal cells will be purified from living embryos using fluorescence activated cell sorting (FACS). RNA isolated from the FACS-purified cells will then be used to construct cDNA libraries. These libraries will be submitted to the NIH-sponsored Washington University EST Center for large scale DNA sequencing analysis and sequence information will be entered into public database directly from the EST center. We will search the database with our EST sequences to """"""""weed out"""""""" those genes already identified. EST sequences whose function is not yet identified will be considered to be """"""""novel"""""""" and will be used to analyze expression patterns and chromosomal locations. Genes identified in these experiments, coupled with the knowledge generated concerning their expression patterns and chromosomal assignment, should serve as excellent candidate genes for the zebrafish embryonic mutations affecting neuronal development.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD039121-02
Application #
6387756
Study Section
Special Emphasis Panel (ZNS1-SRB-P (01))
Program Officer
Henken, Deborah B
Project Start
2000-05-01
Project End
2001-07-31
Budget Start
2001-05-01
Budget End
2001-07-31
Support Year
2
Fiscal Year
2001
Total Cost
$32,880
Indirect Cost
Name
Medical College of Georgia (MCG)
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
City
Augusta
State
GA
Country
United States
Zip Code
30912