Improved and versatile viral 2 A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in C hlamydomonas reinhardtii
Abstract
Summary A significantly improved viral 2A peptide system for dependable high‐level expression of dicistronic genes in Chlamydomonas reinhardtii has been developed. Data are presented demonstrating that use of an especially proficient ‘extended FMDV 2A’ coding region allows production of two independent protein products from a dicistronic gene with almost complete efficiency. Importantly, results are also presented that demonstrate the utility of this 2A system for efficient high‐level expression of foreign genes in C. reinhardtii , which has not previously been reliably achievable in this algal model system. To expand the versatility of the 2A expression system, a number of commonly used selectable marker proteins were assessed for their compatibility with the extended FMDV 2A peptide. Additional experiments demonstrate the feasibility and utility of 2A‐containing dicistronic systems that rely on a strong conditional promoter for transcriptional control and a low‐expression marker gene for selection. This strategy allows easy and efficient delivery of genes of interest whose expression levels require regulation either to mitigate potential toxicity or allow differential expression under controlled experimental conditions. Finally, as an additional practical demonstration of the utility of the extended FMDV 2A system, confocal fluorescence microscopy is used to demonstrate that native and foreign proteins of interestmore »
- Authors:
-
- Department of Biochemistry University of Nebraska Lincoln NE 68588‐0664 USA
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1400704
- Resource Type:
- Publisher's Accepted Manuscript
- Journal Name:
- The Plant Journal
- Additional Journal Information:
- Journal Name: The Plant Journal Journal Volume: 82 Journal Issue: 4; Journal ID: ISSN 0960-7412
- Publisher:
- Wiley-Blackwell
- Country of Publication:
- United Kingdom
- Language:
- English
Citation Formats
Plucinak, Thomas M., Horken, Kempton M., Jiang, Wenzhi, Fostvedt, Jessica, Nguyen, Sanh Tan, and Weeks, Donald P. Improved and versatile viral 2 A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in C hlamydomonas reinhardtii. United Kingdom: N. p., 2015.
Web. doi:10.1111/tpj.12844.
Plucinak, Thomas M., Horken, Kempton M., Jiang, Wenzhi, Fostvedt, Jessica, Nguyen, Sanh Tan, & Weeks, Donald P. Improved and versatile viral 2 A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in C hlamydomonas reinhardtii. United Kingdom. https://doi.org/10.1111/tpj.12844
Plucinak, Thomas M., Horken, Kempton M., Jiang, Wenzhi, Fostvedt, Jessica, Nguyen, Sanh Tan, and Weeks, Donald P. Sat .
"Improved and versatile viral 2 A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in C hlamydomonas reinhardtii". United Kingdom. https://doi.org/10.1111/tpj.12844.
@article{osti_1400704,
title = {Improved and versatile viral 2 A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in C hlamydomonas reinhardtii},
author = {Plucinak, Thomas M. and Horken, Kempton M. and Jiang, Wenzhi and Fostvedt, Jessica and Nguyen, Sanh Tan and Weeks, Donald P.},
abstractNote = {Summary A significantly improved viral 2A peptide system for dependable high‐level expression of dicistronic genes in Chlamydomonas reinhardtii has been developed. Data are presented demonstrating that use of an especially proficient ‘extended FMDV 2A’ coding region allows production of two independent protein products from a dicistronic gene with almost complete efficiency. Importantly, results are also presented that demonstrate the utility of this 2A system for efficient high‐level expression of foreign genes in C. reinhardtii , which has not previously been reliably achievable in this algal model system. To expand the versatility of the 2A expression system, a number of commonly used selectable marker proteins were assessed for their compatibility with the extended FMDV 2A peptide. Additional experiments demonstrate the feasibility and utility of 2A‐containing dicistronic systems that rely on a strong conditional promoter for transcriptional control and a low‐expression marker gene for selection. This strategy allows easy and efficient delivery of genes of interest whose expression levels require regulation either to mitigate potential toxicity or allow differential expression under controlled experimental conditions. Finally, as an additional practical demonstration of the utility of the extended FMDV 2A system, confocal fluorescence microscopy is used to demonstrate that native and foreign proteins of interest bearing post‐translational remnants of the extended FMDV 2A peptide localize correctly to various cellular compartments, including a striking demonstration of the almost exclusive localization of the Rubisco small subunit protein to the pyrenoid of the C. reinhardtii chloroplast in cells maintained under ambient CO 2 concentrations.},
doi = {10.1111/tpj.12844},
journal = {The Plant Journal},
number = 4,
volume = 82,
place = {United Kingdom},
year = {Sat May 09 00:00:00 EDT 2015},
month = {Sat May 09 00:00:00 EDT 2015}
}
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