A Gateway MultiSite recombination cloning toolkit

The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduc...

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Bibliographic Details
Published in:PloS one 2011-09, Vol.6 (9), p.e24531-e24531
Main Authors: Petersen, Lena K, Stowers, R Steven
Format: Article
Language:English
Subjects:
Analysis
Animals
Biology
Caenorhabditis elegans - genetics
Cloning
Cloning vectors
Cloning, Molecular - methods
Deoxyribonucleic acid
Discosoma
DNA
DNA - genetics
DNA, Recombinant - genetics
Drosophila
Drosophila melanogaster
Drosophila melanogaster - genetics
Engineering
Enzymes
Escherichia coli - genetics
Fluorescence
Fragmentation
Gene expression
Genetic Techniques
Genetic Vectors
Genomes
Glycoproteins
Green Fluorescent Proteins - metabolism
Humans
Immunoglobulins
Insects
Ion channels
Microscopy, Fluorescence - methods
Models, Genetic
Muscle function
Neurosciences
Physics
Proteins
Recombination
Recombination, Genetic
Reproducibility of Results
Saccharomyces cerevisiae
Science Policy
Transient receptor potential proteins
Vectors (Biology)
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Description
Summary:The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org).
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0024531