A Novel Method for Mammalian Large Genetic Circuit Assembly and Delivery

Genetic manipulation of mammalian cells provides a foundation for contemporary biological research both basic and applied. Existing methods for construction and introduction of large scale exogenous genetic information into mammalian cells and for creating stable cell lines are not efficient and suffer from limitations in terms of cost, speed, flexibility, and reliability. In this thesis, a novel method is presented for the efficient construction and delivery of complex genetic circuits into mammalian cells. Multi-gene circuits are assembled with high efficiency from a validated modular library into single pieces. The assembled circuits can be used for transient expression and each individual circuit can be integrated into a cellular genome to create a stable cell line. Genetic circuits were constructed that contain several expression units, including inducible control units and fluorescent markers. These circuits were delivered into Human Embryonic Kidney 293 (HEK293) cells for both transient and stable expression cases. Results show that the introduced genetic circuits performed as designed and that stable cell lines, each with the desired phenotype could be created efficiently. Several factors affecting the assembly efficiency and the performance of resulting circuits are also discussed. Thesis Supervisor: Ron Weiss Title: Associate Professor