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Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants
- Source :
- Nature Protocols. 9:1010-1027
- Publication Year :
- 2014
- Publisher :
- Springer Science and Business Media LLC, 2014.
-
Abstract
- Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.
- Subjects :
- Transcriptional Activation
Regulation of gene expression
Ethanol
Models, Genetic
biology
Transgene
Nicotiana tabacum
fungi
food and beverages
Genetically modified crops
biology.organism_classification
Molecular biology
Recombinant Proteins
General Biochemistry, Genetics and Molecular Biology
Viral vector
Cell biology
law.invention
Geminiviridae
Gene Expression Regulation, Plant
law
Tobacco
Gene expression
Recombinant DNA
Transgenes
Subjects
Details
- ISSN :
- 17502799 and 17542189
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- Nature Protocols
- Accession number :
- edsair.doi.dedup.....cf99d4f6ad9d96159303c3888d08ee23