The transcription factor Six1 is overexpressed in numerous types of cancer. Utilizing zebrafish, a better understanding of Six1 function may be achieved through modification of transcriptional co-factors which influence Six1 function, specifically Eya2 and Eya3. To decrease expression of these proteins, morpholinos targeting Eya2 or Eya3 were injected into zebrafish embryos. Determination of morpholino function was undertaken to confirm research methods are sound. Our hypothesis was that Eya2 and Eya3 morpholinos may do one of two things, or both; morpholinos could cause degradation of target mRNA, or they will bind to immature RNA and cause exon skipping, and thus a non-functional mRNA will be produced. To test this, RNA was isolated 24 hours after embryos were injected with morpholinos, which was then used for the synthesis of cDNA. The cDNA was then amplified through either PCR or qPCR specific to the Eya exon regions of interest. Agarose gel analysis of PCR product revealed a lower amount of both Eya2 and Eya3 mRNA in the morphants injected with morpholino, as well as an alternate, smaller product resulting from exon skipping. qPCR indicated a lower amount of Eya2 mRNA as well. Overall, these results indicate that the morpholinos are functioning as intended.
