Date of Award

6-2020

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Bournias-Vardiabasis, Nicole

Abstract

Neuroblastoma (NB) is an extracranial tumor that affects the nervous system and accounts for approximately 650 – 800 cases diagnosed per year in children under the age of 5 (www.cancer.org/cancer/neuroblastoma/about/key-statistics.html). NB occurs at a relatively rare rate of 10.2 per million children under 15 years old but accounts for 12-15% of pediatric cancer death because patients with high-risk NB (HRNB) have 5-year survival rates between 40-50%(Brodeur, 2003; Park et al. 2010). Developmental biology research has recognized neural crest cells (NCC) as the progenitor cells of NB, as tumors form in the trunk NC derived sympathetic nervous system (Dupin, 2013; Simões-Costa et al., 2015). Genome-wide association studies (GWAS) of these tumors reveal several chromosomal aberrations and genes related to the development of NB (Pugh et al., 2013; Seeger et al., 1985). Unfortunately, the heterogeneity of the disease has revealed a low mutation frequency in suspected oncogenes, along with an assortment of chromosomal gains and losses, resulting in a variety of mechanisms of NB formation. These structural features demonstrate the complexity of NB tumorigenesis and the unlikeliness of developing a single target treatment (Matthay, K et al., 2012). Amongst all the predictors recognized in NB formation, amplification of the MYCN gene occurs in 20% of tumors and serves as the most significant prognosticator of poor outcomes in HRNB patients (Brodeur, 1984). These insights have resulted in NCC experiments in mice, zebrafish, and primary cell models to study NB development under the overexpression of MYCN and other oncogenes such as ALK and LMO1(Weiss et al.,1997; Zhu et al., 2012; Olsen et al., 2017). While these models have elucidated the association of MYCN and LMO1 overexpression in NCC to NB formation, they lack a direct descriptive model of NB formation in human NCC.

Building off the current understanding of genes synergistically driving neuroblastoma, I utilized H1 human embryonic stem cells (hESC) to create a cell line inducible for LMO1 and MYCN. Through genetic engineering, and subcloning, I created two constructs: a plasmid containing the MYCN gene under the regulation of the Tetracycline On (TET-ON) system and the LMO1 construct under the regulation of TET-ON. I sequenced the constructs, analyzed their genetic composition, and found the TET-ON LMO1 suitable for integration into the H1 hESC. After nucleofection and genetic screening, the data suggest the production of a mixed population of H1 hESC containing a subset of cells that appear to have integrated the TET-ON LMO1 into the AAVS1 locus at chromosome 19. Further attempts are required for the complete development of the H1 hESC TET- 3G LMO1 plus MYCN inducible cell line.

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