ArticleNucleic acids research2013
Modular assembly of transposon integratable multigene vectors using RecWay assembly.
Article in Nucleic acids research, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed.
- Article
- Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.Molecular therapy. Methods & clinical development · 2020Article
- Development of innate immune cells from human pluripotent stem cells.Experimental hematology · 2019Review
- Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity.Cell stem cell · 2018Article
- Concise Review: Human Pluripotent Stem Cells to Produce Cell-Based Cancer Immunotherapy.Stem cells (Dayton, Ohio) · 2018Review
- Article
- piggyBac-ing models and new therapeutic strategies.Trends in biotechnology · 2015Review
- Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.Scientific reports · 2014Article
- Trp53 haploinsufficiency modifies EGFR-driven peripheral nerve sheath tumorigenesis.The American journal of pathology · 2014Article
- Mouse models of cancer: Sleeping Beauty transposons for insertional mutagenesis screens and reverse genetic studies.Seminars in cell & developmental biology · 2014Review
- HomeRun Vector Assembly System: a flexible and standardized cloning system for assembly of multi-modular DNA constructs.PloS one · 2014Article
- Simple and efficient methods for enrichment and isolation of endonuclease modified cells.PloS one · 2014Article
Corrections and comments
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Authors and funding
9 authors.
Funding
Abstract
Studying complex biological processes such as cancer development, stem cell induction and transdifferentiation requires the modulation of multiple genes or pathways at one time in a single cell. Herein, we describe straightforward methods for rapid and efficient assembly of bacterial marker free multigene cassettes containing up to six complementary DNAs/short hairpin RNAs. We have termed this method RecWay assembly, as it makes use of both Cre recombinase and the commercially available Gateway cloning system. Further, because RecWay assembly uses truly modular components, it allows for the generation of randomly assembled multigene vector libraries. These multigene vectors are integratable, and later excisable, using the highly efficient piggyBac (PB) DNA transposon system. Moreover, we have dramatically improved the expression of stably integrated multigene vectors by incorporation of insulator elements to prevent promoter interference seen with multigene vectors. We demonstrate that insulated multigene PB transposons can stably integrate and faithfully express up to five fluorescent proteins and the puromycin-thymidine kinase resistance gene in vitro, with up to 70-fold higher gene expression compared with analogous uninsulated vectors. RecWay assembly of multigene transposon vectors allows for widely applicable modelling of highly complex biological processes and can be easily performed by other research laboratories.
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