Developing a System to Arrest Transformation using a Zebrafish Model for Melanomagenesis

The recent emergence of RNA-guided CRISPR-Cas9 gene editing system adapted from the natural defense mechanism found in bacteria and archaea has made the manipulation of genetic loci more feasible than ever. Using this technique we attempted to eliminate a target sequence of about 20 nucleotides from the promoter of the protein ICER (Inducible cAMP Early Repressor) located just upstream of the start sequence. ICER is a small transcription factor and supposed tumor suppressor protein that comes from the CREM (cAMP Responsive Element) gene. ICER has been found to be absent in tumor cells, marked for degradation by the ubiquitin-proteasomal pathway. By eliminating the target sequence we hope to knockout functional ICER protein in order to observe any possible effects this could have. Essentially it is believed that the elimination of ICER would lead to the generation or acceleration of tumor development. To achieve this we are using the well-characterized zebrafish (Danio rerio) melanoma model as a paradigm. Two types of methods are being utilized to insert our Cas9/gRNA construct into zebrafish: transfection via PAC2 cells and direct injection into one-cell stage embryos. In addition, we are using a number of techniques including PCR amplification, sequencing, T7 endonuclease assay, and TOPO cloning in order to provide evidence of target sequence manipulation. The long-termed objective of this study will be to determine whether eradication of ICER will affect the tumorigenesity of wild-type (EK) zebrafish in comparison to the established zebrafish model for melanoma