Basic Gateway principles

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NB: The Tol2kit uses three-insert multisite Gateway cloning (att4-att1-att2-att3) from Invitrogen. It is not easily compatible with the newer "Gateway Pro" system, for which the outermost sites are att1-att2.

[edit] Principles of Gateway recombination

Here's a diagram of a Gateway BP reaction, showing an example of how a "middle" clone (pME) is constructed using PCR with gene-specific primers to add att sites, then recombination with a donor vector.

BP reaction

Here's a diagram of a three-insert multisite Gateway LR reaction, combining three entry clones (p5E, pME, p3E) with the destination vector pDestTol2pA.

LR reaction

[edit] How we use this in the Tol2kit

To generate zebrafish expression constructs, we generally place enhancer-promoter constructs in the 5' (p5E) clones, reporters or genes of interest in the middle (pME) clones, and polyadenylation signals, C-terminal fusion tags, or IRES tags in the 3' (p3E) clones. Of course there are other possibilities as well, e.g. putting an N-terminal fusion tag in the middle clone and then a gene of interest in the 3' clone.

The destination clone bears transposon ends (Tol2 terminal inverted repeats), a polyadenylation signal (in case no polyA signal is included in the 3' clone), and sometimes a transgenesis marker like cmlc2:egfp-polyA.

Given the different clones in this toolkit, one can thus add a great deal of functionality to an expression clone by assembling it using a multisite Gateway reaction. For instance, by using p5E-bactin2, pME-nlsmCherry, p3E-IRES-EGFPCAAX-pA, and pDestTol2CG2, a single Gateway reaction can yield the construct bactin2:nlsmCherry-IRES-EGFPCAAX-pA; cmlc2:egfp-pA, which will express nuclear mCherry and membrane-localized EGFP ubiquitously, along with cytoplasmic EGFP in the heart as a transgenesis marker.

In the future, we are planning to build new destination vectors to use other transposons, e.g. Sleeping Beauty, and to add other desired elements such as insulators.

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