Extended transient transfection by repeated delivery of in vitro-transcribed RNA
Name
435528760-MIT.pdf
Description
Full printable version
Size
24.71 MB
Format
Adobe PDF
Checksum (MD5)
8f34c55d2374c96f3053976d828d0cdc
Author(s)
Angel, Matthew
Advisor(s)
Mehmet F. Yanik.
Date Issued
2008
Publisher
Massachusetts Institute of Technology
Abstract
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.
Description
Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.
Includes bibliographical references (p. 53-56).
Subjects
Electrical Engineering and Computer Science.
MIT Department
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
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