Most genetic manipulations involve inserting "foreign" pieces of DNA into the genome of a manipulated organism. The means to do this is mostly a small piece of circular DNA called a plasmid or vector. Making these vectors in the laboratory in a "classical" way (cutting and ligating DNA required using enzymatic reactions) is labor intensive, time-consuming, and unsuitable for high throughput cloning. The means to solve this problem is with the Gateway cloning system that will perform all the necessary cuts and ligations in a fast and accurate biochemical reaction called site-specific recombination.
Gateway technology provides a way to move genes into multiple vector systems for functional analysis and protein expression (Invitrogen, Gateway Technology Manual, 2003). The Gateway cassette (GW) is a unique sequence, cloned into the destination plasmid, while the desired gene is cloned into the entry vector. The gene in the incoming plasmid (which has kanamycin or zeocin resistance) is flanked by the attl site, whereas the Gateway cassette in the destination plasmid (which has ampicillin resistance) is enclosed by the attR sites. These sites will rejoin and swap the order in the attL site and attR if the clonase is added (LR reaction) (Invitrogen, Gateway Technology Manual, 2003). Following the LR reaction protocol between the entry and the destination vector, the desired gene is inserted in the Gateway cassette holder. The Gateway cassette also contains chloramphenicol resistance genes. During the LR reaction, the chloramphenicol resistance gene is replaced by the desired gene, which makes it possible to calculate the resulting clones produced with chloramphenicol (the right target clone will not stand chloramphenic but holds only ampicillin).
Furthermore, Invitrogen (TM) has introduced the new MultiSite GatewayÃ,® multi-tape platform, enabling the insertion of up to four DNA elements into each destination vector of Gateway®.
Video Gateway cassette
References
Gateway Technology Manual, a universal technology for cloning DNA sequences for functional analysis and expression in systems. September 22, 2003. Introduction. pp 1-7. Invitrogen Life Technology.
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