Molecular Techniques Facility
The Molecular Techniques Facility provides Center members with access to state-of-the-art equipment and methodology and the expert personnel required for the successfully completion of complex toxicogenomic experiments with aquatic organisms.
More specifically, the core provides focused, specialized support in the development and dissemination of techniques for the modulation and measurement of gene expression in zebrafish. As such, the core strives to accomplish its missions by:
Providing access to state-of-the-art equipment for doing molecular biology and genomic experiments.
Offering expert technical advice in experimental design and implementation, and data interpretation.
Continually seeking out educational opportunities for learning new techniques.
Adapting molecular biology based techniques from other model systems for use in aquatic species.
Developing novel techniques and technologies for use in zebrafish, including: development of improved means for creating transgenic zebrafish, involving design and construction of expression vectors. And development of vector based RNAi technology for knocking out expression of specific genes in zebrafish, from embryonic to adult expressed genes.
If you are interested in learning more about the Molecular Techniques Facility, please contact Dr. Henry Tomasiewicz (414) 382-1746.
In addition to access to state of the art equipment Center investigators benefit from having access to the latest techniques and technologies for not only analyzing gene expression levels, but for either augmenting gene expression through the use of transgenic fish or reducing a genes expression via different anti-sense strategies.
At the genomic level, the Core has the capability to do High Throughput PCR based genomic screens using DNA Tetrad Engines equipped with 384 well thermal cycling blocks to amplify cDNA clones that are spotted onto microarray slides, probed and analyzed for changes in gene expression caused by exposure to an environmental toxicant using a slide reader and in house developed bioinformatics programs.
Transgenic zebrafish expressing fluorescent proteins or tox gene encoded proteins fused in frame with fluorescent protein from tissue specific promoters can be used to look at the effects of environmental toxicants on zebrafish at the Cellular or Tissue level.
Targeted knockdown of gene expression in embryonic and early larval stages of zebrafish can be accomplished using anti-sense morpholino oligonucleotides. Using this approach the effects on zebrafish development of knockdowns of specific tox genes on exposure to environmental toxicants can be ascertained.
Similarly, using vector based RNA interference, knockdown of target genes lasting into adulthood in zebrafish are used to examine the long term effects of toxicant exposure on fish deficient in toxicologically relevant genes.
The Center has also developed a line of zebrafish, called sheer, that are transparent throughout adulthood. The Sheer line of fish were generated by intercrossing three different mutant lines of zebrafish, each defective in a different pigment, until the resulting transparent fish was obtained.