Date of Award
Master of Science (MS)
Chemical and Biomolecular Engineering
Mark A Blenner
Mammalian artificial chromosomes, or MACs, have been studied as a potential avenue for hosting large numbers of transgenes in mammalian cells. MACs have several advantages over viral-based methods for transgene expression, including a lack of limits on loading capacity, which bypasses issues associated with integration into the genome. One area of research in which MACs can be applied is the biomanufacturing of protein-based therapeutics, where reported genome instability in Chinese hamster ovary (CHO) cells can lead to reduced product titer. MACs can potentially aid in solving this issue by providing alternate hosting sites for transgenes for integration of protein-based therapeutic production. However, some hurdles exist in the path of utilizing MACs as a biology tool, including the acquisition of sufficient mass and concentration of a MAC, the molecular cloning of a transgene into a MAC, and delivery of the cloned MAC to target mammalian cells. To address this, improvements were made at the steps of transformation of the MAC into E. coli, isolation of positive colonies, and subsequent kit purification to generate sufficient masses and concentrations for downstream applications. Using Gibson Assembly, a selectable marker, glutamine synthetase (GS), was successfully cloned onto the MAC, yielding the construct MAC-GS. MAC-GS was subsequently electroporated into suspension CHO cells, and selection by removal of L-glutamine demonstrated the functionality of GS. These results represent a positive step forward for the implementation of MACs as a useful synthetic biology tool.
Wang, Charles, "Mammalian Artificial Chromosomes as a Synthetic Biology Tool for Transgene Expression" (2019). All Theses. 3233.