InSITE Donor Cassettes

The InSITE (Integrase Swappable In Vivo Targeting Element) system allows for conversion of a GAL4 enhancer trap into one containing lexA, QF, or a number of other transcriptional regulators using simple genetic crosses. Initially, GAL4-bearing enhancer trap lines are isolated by either P- or piggyBac-based element transposition (two suitable “ammunition” lines that can be used as transposition sources are available). In subsequent crosses, the GAL4 cassette in these lines can then be swapped for “donor” cassettes encoding other genetic effectors. 

The InSITE system is described in Gohl et al. 2011. “A versatile in vivo system for directed dissection of gene expression patterns.” Nature Meth. 8(3):231-237. FBrf0213241 

Gohl et al. Supplementary Figure 5 containing crossing schemes for doing swaps can be found here

A list of currently available InSITE GAL4 lines can be found here.

insitev41

In the presence of an enhancer trap insertion (the “target”), the donor insertion, and FLP and phiC31 recombinases, FLP recombinase will FLP-out the FRT-flanked attB-bearing donor and phiC31 will put the freed donor into the attP site of the enhancer trap target. At this step, the target site (which now carries both GAL4 and the donor gene) contains a FRT site which can be used for generating deletions. Subsequent steps using Cre recombinase complete the swap by removing the GAL4 (this step also removes the FRT).


Donor cassettes (see "Donor stocks" below for lines carrying these insertions):
P{ID.GAL4AD}GAL4-activator domain fusion gene for the split GAL4 system
P{ID.GAL4DBD}GAL4-DNA binding domain fusion gene for the split GAL4 system
P{ID.GAL80}GAL80 (GAL4 inhibitor)
P{ID.LexA}LexA transcriptional activator
P{ID.QF}QF transcriptional activator
P{ID.VP16AD}GAL4-VP16 activator domain fusion gene for the split GAL4 system