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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Electrical Engineering and Computer Science.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2012

Author dc:creator

• Li, Yinqing, Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Ron Weiss.

• Electrical Engineering and Computer Science.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/71499

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/71499