Gateway Cassette

Genetic manipulation mostly involves inserting a "foreign" piece of DNA into the genome of the manipulated organism. The means to do this is mostly a small piece of circular DNA called plasmid or vector.Creating these vectors in the lab by "classical" means (= cutting and ligation of the required dna by enzymatic reactions) is labor intensive/time consuming and not suited for high throughput cloning. A means to solve this issue is by the Gateway cloning system which will perform all the needed cutting and ligation in one fast and accurate biochemical reaction, named site specific recombination.

Details

The Gateway Technology provides a quick and highly efficient way to move genes into a multiple vector system for functional analysis and protein expression (Invitrogen, Gateway Technology Manual, 2003). The Gateway Cassette (GW) is a unique sequence, which is cloned into a destination plasmid, whereas the desired gene is cloned into the entry vector. The gene in the entry plasmid (which possesses kanamycin or zeocin resistancy) is flanked by attL sites, whereas the Gateway cassette in the destination plasmid (which possesses ampicillin resistancy) is flanked by attR sites. These sites will recombine and exchange the sequences within the attL and attR sites if clonase is added (LR reaction) (Invitrogen, Gateway Technology Manual, 2003). Following the LR reaction protocol between the entry and the destination vectors, the desired gene is inserted in place of the Gateway cassette. The Gateway cassette also contains a chloramphenicol resistance gene. During the LR reaction, the chloramphenicol resistance gene is replaced by the desired gene, which makes it possible to counterselect the resulting destination clone with chloramphenicol (correct destination clones will not be chloramphenical resistant but only ampicillin resistant).

A big advantage of this technology is that it allows putting the gene of interest (from the entry vector) into many different destination vectors by only one LR reaction.

References

Gateway Technology Manual, a universal technology to clone DNA sequences for functional analysis and expression in multiple systems. 22 September 2003. Introduction. pp 1–7. Invitrogen Life Technologies.