Figure 1.

Zinc-finger nuclease (ZFN) linker variants. (a) Schematic of the EB2-N homodimer. All ZFN variants are based on EB2-N²³ and contain an N-terminal HA epitope tag, three zinc-fingers (1, 2, 3), the inter-domain linker, and the FokI cleavage domain. The two target half-sites (L and R) are separated by a spacer sequence of defined length (see Supplementary Table S1). (b) Sequence of ZFN linker variants. The names, linker lengths, and the relevant amino acid sequences are indicated. The ZFN inter-domain linker length has been defined as the number of amino acids (aa) between the last conserved histidine in the third zinc-finger (F3) and the first residue of the FokI cleavage domain (QLV). Note that in some variants the first residues of the FokI domain were deleted and offset against the linker length. (c) Expression analysis of ZFN linker variants. HEK293T cells were transfected with an expression plasmid encoding a ZFN linker variant along with pEGFP as an internal transfection control. After 30 h, the cells were harvested and the lysates were probed simultaneously with antibodies against both the HA tag and EGFP. HA-tagged I-SceI and mock-transfected cells (cto) were included as controls. The positions of the various ZFNs, I-SceI, EGFP, and size markers are indicated.