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Site-Specific Incorporation of Unnatural Amino Acids into Receptors Expressed in Mammalian Cells

Monahan, Sarah L. and Lester, Henry A. and Dougherty, Dennis A. (2003) Site-Specific Incorporation of Unnatural Amino Acids into Receptors Expressed in Mammalian Cells. Chemistry and Biology, 10 (6). pp. 573-580. ISSN 1074-5521. doi:10.1016/s1074-5521(03)00124-8. https://resolver.caltech.edu/CaltechAUTHORS:20200427-134043259

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Abstract

We describe an approach to achieve unnatural amino acid incorporation into channels and receptors expressed in mammalian cells. We show that microelectroporation provides a general method to deliver DNA, mRNA, and tRNA simultaneously. In both CHO cells and cultured neurons, microelectroporation efficiently delivers an in vitro transcribed, serine amber suppressor tRNA, leading to nonsense suppression in a mutant EGFP gene. In CHO cells, both natural and unnatural amino acids chemically appended to a suppressor tRNA are site specifically incorporated into the nicotinic acetylcholine receptor (nAChR). Electrophysiology confirms the expected functional consequences of the unnatural residue. The microelectroporation strategy described here is more general, less tedious, and less damaging to mammalian neuronal and nonneuronal cells than previous approaches to nonsense suppression in small cells and provides the first example of unnatural amino acid incorporation in mammalian cells using chemically aminoacylated tRNA.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/s1074-5521(03)00124-8DOIArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 2003 Cell Press. Published by Elsevier Ltd. Received 2 April 2003, Revised 8 May 2003, Accepted 13 May 2003, Available online 24 June 2003. We thank Jack Horne, Gabriel Brandt, and Raad Nashmi for their helpful advice and Mike Walsh (electronic shop) and Herb Adams (machine shop) for their assistance. Supported by NIH (NS-34407, NS-11756, GM-29836). S.L.M. held an NSERC (PGS-B) scholarship and was a fellow of an NIH Predoctoral Trainee Grant (GM-08501).
Funders:
Funding AgencyGrant Number
NIHNS-34407
NIHNS-11756
NIHGM-29836
Natural Sciences and Engineering Research Council of Canada (NSERC)UNSPECIFIED
NIH Predoctoral FellowshipGM-08501
Issue or Number:6
DOI:10.1016/s1074-5521(03)00124-8
Record Number:CaltechAUTHORS:20200427-134043259
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200427-134043259
Official Citation:Sarah L Monahan, Henry A Lester, Dennis A Dougherty, Site-Specific Incorporation of Unnatural Amino Acids into Receptors Expressed in Mammalian Cells, Chemistry & Biology, Volume 10, Issue 6, 2003, Pages 573-580, ISSN 1074-5521, https://doi.org/10.1016/S1074-5521(03)00124-8. (http://www.sciencedirect.com/science/article/pii/S1074552103001248)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102813
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:27 Apr 2020 20:50
Last Modified:16 Nov 2021 18:15

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