Zinc-finger Nuclease Stocks

Zinc-finger nucleases create double-stranded DNA cuts at specific sites based on dimerization of the FokI endonuclease. Each nuclease monomer is localized via zinc-finger motifs binding to genomic sequences flanking the cleavage site as shown below for monomers directed to the yellow gene.

Figure from Beumer et al. (2006). Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172: 2391-2403 depicting zinc-finger nuclease cleavage of the yellow gene and its repair using a transgene template.

The figure also shows the structure of a repair template used to introduce a new sequence into the yellow gene. This template can be excised in vivo from the transgene P{y[M.donor]} by FLP recombinase and either used in a circular form or linearized by the I-SceI endonuclease.

The following table shows stocks expressing zinc-finger nuclease monomers for cleavage of yellow. It also shows a stock with P{y[M.donor]}.

Stock #Genotype
64316v[1]; P{ry[+t7.2]=hs-yA}20b, P{ry[+t7.2]=hs-yB}7a; ry[506]
64270w[*]; P{w[+mC]=hs-yA.P501T.I538K}2C/SM6a
64271w[*]; P{w[+mC]=hs-yB.Q486E.P501T}2C/MKRS

The following table shows similar stocks for cleavage of the rosy gene.

Stock #Genotype
64273w[*]; P{w[+mC]=hs-ryA}1C/SM6a
64274w[*]; P{w[+mC]=hs-ryB}2C/TM3, Sb[1]
64275w[*]; P{w[+mC]=UAS-ryA}14C, P{w[+mC]=UAS-ryB}1B/SM6a

Experiments involving cleavage of yellow and rosy by zinc-finger nucleases and their repair with template constructs are described in Beumer et al. (2006). "Efficient gene targeting in Drosophila with zinc-finger nucleases".