Genome Engineering of Mammalian Cells using Phage N15 Protelomerase

TelN protelomerase is a multi-functional microbial enzyme that functions only on telomere-occupancy-site (tos) in constructing linear DNA. While TelN-tos-based linear DNA is stably maintained in bacteria, its stability in the mammalian cell remains unclear. The thesis examines TelN’s stable functionalities in human and mouse cells to process and maintain DNA-bearing tos. It was found that TelN’s cut, rejoin and replicating activities are significantly retained in tested cells and impact on linear DNA formation. Overall, the study describes useful methodologies and approaches to investigate the stable native functions of the microbial genetic system to form linear DNA in animal cell environment.