Date of Award
Master of Science
Genetics, Development and Cell Biology
Genetics and Genomics; Molecular, Cellular and Developmental Biology
Transposon somatic mutagenesis is a forward genetics approach that can be used to identify novel genetic drivers of cancer. This research has developed tools required for performing these transposon mutagenesis screens in zebrafish. Transgenic zebrafish lines were created that allow for tissue-specific and inducible expression of the transposase required for the Sleeping Beauty (SB) transposon system. Mutagenesis screens will be performed in a pRB deficient background, which gives rise to highly proliferative and undifferentiated tumors resembling primitive neuroectodermal tumors (PNETs). This could reveal cooperating genes that interact with rb1 to affect brain cancer. To this end, neural progenitors were targeted using the Tg(krt5:SB11) transgenic line. The ability of this transgenic to mobilize transposons was demonstrated in whole larvae and adult brains. Also, an inducible transposase source in the Tg(ubi:floxed-SB11) transgenic line was able to increase transposon mobilization in response to Cre injection, despite a low level of background expression. Other tissue-specific promoters were explored, including gfap and nestin, which may result in increased tumorigenesis.
Several mutagenesis screens are currently being monitored in both wild type and tumor susceptible backgrounds. Common integration sites in the zebrafish genome are also being mapped to aid in the analysis of data from these screens by removing background that does not contribute to tumorigenesis. A small scale mutagenesis screen in a wild type background has resulted in a single fish out of ten that developed a brain tumor after 8 months of age. Importantly, analysis showed that transposon mobilization was specific to tumor tissue, demonstrating that the Tg(krt5:SB11) transgenic line can target mutagenesis to specific cell types. The results of these transposon mutagenesis screens could lead to a better understanding of the genetics behind PNETs and open possibilities for new treatments.
Mauldin, Joshua, "The development of tools for transposon somatic mutagenesis in zebrafish" (2016). Graduate Theses and Dissertations. 15768.