Extended transient transfection by repeated delivery of in vitro-transcribed RNA

An experimental study was performed to evaluate a novel method of controlling protein expression by repeatedly transfecting cells with in vitro-transcribed RNA. Transcripts encoding six factors, each known to play an important role in cell-type specification and maintenance, were designed and synthesized. Aspects of the design were optimized, and the intracellular stability and translation efficiency of the ivT RNA were quantified. Transcripts were delivered to cells by electroporation, and a method of increasing the rate at which cells recover from ivT-RNA transfection by combined knockdown of innate-immune-related genes was developed. Using this technique a high, approximately steady-state level of protein expression was transduced in MRC-5 human fetal-lung fibroblasts by repeated transfection with capped, poly(A)+ ivT RNA encoding a protein with an intracellular half-life of approximately three days. Transfection at 24-hour intervals with ivT RNA encoding a less stable protein resulted in protein expression that peaked twelve hours after each transfection, and diminished before the next transfection. In both cases, cells sustained a high rate of proliferation. In this study, extended transient transfection by repeated delivery of ivT RNA was shown to transduce expression of defined factors in cultured cells without genetic modification or the extensive screening required in small-molecule approaches and with significant control over the level of transduced protein. This technique may become a powerful tool in the development of new directed-differentiation and cell-type-conversion protocols.

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

2008

Author dc:creator

• Angel, Matthew

Advisor dc:contributor.advisor

• Mehmet F. Yanik.

• 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/1721.1/47895

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/47895