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Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
Modular assembly of transposon integratable multigene vectors using RecWay assembly
Branden S Moriarity1, Eric P Rahrmann, Vincent W Keng
1Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu
Nucleic Acids Research
|February 28, 2013
Summary
Researchers developed RecWay assembly for efficiently creating multigene vectors. This method enables better modeling of complex biological processes like cancer development and stem cell research by improving gene expression.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Gene Regulation
Background:
- Complex biological processes necessitate simultaneous modulation of multiple genes or pathways within single cells.
- Existing methods for assembling multiple genes into vectors can be inefficient or lack modularity.
Purpose of the Study:
- To develop a straightforward, rapid, and efficient method for assembling bacterial marker-free multigene cassettes.
- To create modular multigene vectors that can be randomly assembled into libraries.
- To enhance the stable integration and expression of multigene vectors using insulator elements and the piggyBac system.
Main Methods:
- RecWay assembly, utilizing Cre recombinase and the Gateway cloning system, for multigene cassette assembly.
- Generation of randomly assembled multigene vector libraries using modular components.
- Integration and excision of multigene vectors via the piggyBac (PB) DNA transposon system.
- Incorporation of insulator elements to prevent promoter interference in stably integrated vectors.
Main Results:
- Demonstrated successful assembly of up to six complementary DNAs/short hairpin RNAs into bacterial marker-free multigene cassettes.
- Developed randomly assembled multigene vector libraries.
- Showcased stable integration and expression of insulated multigene piggyBac transposons containing up to five fluorescent proteins and a resistance gene.
- Achieved up to 70-fold higher gene expression in insulated vectors compared to uninsulated controls due to prevention of promoter interference.
Conclusions:
- RecWay assembly provides a versatile and efficient platform for constructing multigene vectors.
- Insulated multigene piggyBac transposons offer significantly improved and stable gene expression for complex biological modeling.
- The described methods are widely applicable for modeling complex biological processes and are readily adoptable by other research laboratories.
