Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


LIN

Identification and characterization of tissue-specific genes using transgenic zebrafish

Shuo Lin

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA

It is important that an Expressed Sequence Tag (EST) database produced for a model organism represents mRNA transcripts from all possible tissues. Zebrafish has become an important model organism for genetic studies of vertebrate development and an EST database is currently being generated. Tissue-specific cDNA libraries are usually made from surgically excised organs of larger animals but this technique is not applicable for the construction of tissue- or cell lineage-specific cDNA libraries from zebrafish embryos due to their extremely small size.  To overcome this problem, we developed transgenic zebrafish that differentially express the green fluorescent protein (GFP) reporter gene.  These fish allow the isolation and purification of lineage-specific embryonic cells using fluorescence activated cell sorting (FACS).  To date, we have generated transgenic zebrafish that express GFP in embryonic erythroid, lymphoid, olfactory, pancreas, brain and neuronal progenitor cell lineages.  RNA has been isolated from the FACS-purified GFP positive cells and used to construct cDNA libraries for the EST production.  Using the ESTs from these cDNA libraries, we have also initiated a large-scale whole mount RNA in situ screen to identify novel genes that are expressed in specific tissues. Finally, we have utilized transgenic zebrafish to characterize function of tissue-specific genes and the study of a novel hematopoietic TNF receptor will be presented.

 


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