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The Development of Lung Adenocarcinoma Model Using Transgenic Oncopigs
thesisposted on 2021-05-01, 00:00 authored by Yifan Wang
This study aims to develop a large animal model of lung adenocarcinoma using Oncopigs. Oncopigs are genetically engineered to harbor a heterozygous transgene encoding TP53R167H and KRASG12D under Lox-STOP-Lox control. Expression of the oncogenes can be switched on by introducing Cre recombinase in targeted cells. Lung tissue was harvested from Oncopigs, and primary small airway epithelial cells were enriched via antibody-mediated magnetic bead selection. The primary Oncopig small airway epithelial cells were cultured to reach confluence prior to introducing genetic perturbations in an effort to achieve oncogenic transformation. Overexpression of TP53R167H and KRASG12D encoded by the Oncopig transgene in small airway epithelial cells lead to cell death characterized by the accumulation of large cytoplasmic vesicles. The intracellular vesicular accumulations were confirmed to be derived from pinocytosis by a dextran incorporation assay, which highlights this particular form of cell death as methuosis. Oncogenic transformation in pig lung small airway epithelial cells cannot be achieved by only expressing the Oncopig transgene encoding TP53R167H and KRASG12D. Lentiviral-mediated overexpression of various cell cycle regulators was explored in an effort to overcome methuosis associated with expression of the Oncopig transgene. Oncopig small airway epithelial cells transduced with CDK4, hTERT and CCND1 survived and expanded upon Cre-induced expression of TP53R167H and KRASG12D for over four months, in addition to cells transduced with only one lentiviral vector encoding CDK4R24L.