Abstract
Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.
Highlights
Most contemporary investigations of cellular and molecular processes necessitate the use of synthetic DNA vectors
We previously showed that chained enhanced SIBR (eSIBR) artificial microRNA (amiRNA) expressed from lentiviral vectors potently knocked down Cadm1-3 in cultured rat hippocampal neurons when cells were infected at saturating titers [30]
We developed a 3’ entry vector, p3E-SGTAP, based on an optimized tandem affinity purification (TAP) method that is amenable to much smaller amounts of starting material [44]
Summary
Most contemporary investigations of cellular and molecular processes necessitate the use of synthetic DNA vectors. Recombinant cloning of plasmid vectors is the most commonly used method for transgenic analyses. After the first successful demonstration of gene expression from exogenous DNA in mammalian cells [1], synthetic vectors were established as a powerful method to assay gene function in vitro and in vivo. The development of sophisticated techniques such as genetic knockdown and knockout allowed more intricate and detailed investigations. The continued advancement of recombinant DNA technologies has provided the modern biologist with an arsenal of molecular tools. Use of these techniques, often requires the laborious construction and validation of complex, multi-component vectors
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