Description:

Assigning genes to functional behavior is one of the challenging tasks in the post genomic era. To date, gene silencing methods in zebrafish only allowed studying functions of genes that are involved in very early, primarily embryonic development. The overall objective of our project is to develop an effective and specific gene silencing technology to elicit the function of olfactory receptors with the long term goal to understand the genetic background of olfactory learning, memory and imprinting; specifically, how exposure to specific odors during early development shapes future nutritional and social preferences.

The recent discovery that small interfering RNA (siRNA) introduced into the cells selectively alters endogenous expression of a target gene, has become the most powerful technique to study gene function. Instead of introducing foreign DNA to generate knockout animals or plants, which leads to undesirable hereditary changes, the ability to knockdown the specific mRNA is very valuable. This is accomplished by the introduction into the cells of a 21-mer oligonucleotide complementary to the mRNA.

We have chosen a transgenic fish which expresses a fusion of a histone variant promoter, H2A.F/Z, to the green fluorescent protein (GFP). Therefore, every cell in this animal is highly fluorescent. The introduction of a siRNA specific for the mRNA of GFP should decrease the expression of that protein. We have established that the imaging systems available through the BioCurrents Research Center can quantitate the changes in fluorescence of each specimen during development. This allows us to measure the correlation between the concentration of siRNA used and the expression of the GFP gene in the animal.

Progress:

To evaluate the time course of gene knock down by dsRNA in zebra fish, we experimentally injected eggs of zebrafish expressing a gene for enhanced yellow fluorescent protein (pE-YFP) with dsRNA against the YFP gene. DsRNA for the pE-YFP gene evoked a decrease (35%) in yellow fluorescence with the onset of silencing of YFP expression. We determined the time course of gene silencing by measuring fluorescence in control and dsRNA injected larvae.
Our experiments show for the first time that injections of dsRNA can knock down genes for 15 days; much longer than other gene silencing methods, such as morpholinos.

The continued work will open new exciting possibilities in zebrafish research to understand the genetic background of sensory processing.