1. Custom Zinc-Finger Nucleases for Use in Human Cells
Stephen Alwin, Maja B. Gere, Eva Guhl, Karin Effertz, Carlos F. Barbas, David J. Segal, Matthew D. Weitzman, Toni Cathomen 
Molecular Therapy 
Volume 12, Issue 4, Pages (October 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Analysis of DNA binding. (A) Sequence motifs. The sequences of the crucial residues of the zinc-finger modules and the corresponding DNA-binding sites are shown. Module “A” was designed to recognize DNA triplet “a” and so on. (B) Schematic. Custom transcription factors (TFs) consist of an HA tag (black box), the VP16 transcriptional activation domain (AD), the SV40 NLS (n), and three zinc-finger modules. The reporter plasmids contain the luciferase gene, a minimal promoter element, and upstream binding sites for the custom TFs (xx). Each x represents a 9-bp recognition motif as defined in (A). (C) Reporter assay. 293T cells were transfected with expression plasmids for the custom TFs and reporter plasmids containing binding sites for GZF1 and GZF2 (1-2/Luc) or GZF3 and GZF4 (3-4/Luc). The graph displays luciferase activity normalized for transfection efficiency and relative to transfection with empty vector (AD). **Statistically significant (P < 0.01). (D) Western blot. Transfected 293T cells were harvested after 40 h and cell lysates probed with an anti-HA antibody. AD-ZF5C is a control zinc-finger protein, while “cto” marks a negative control.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Design and expression kinetics. (A) Design. All ZFNs contain an HA tag (black box), three zinc-finger modules, and the nuclease domain of FokI (Fok1-N). GZF3n-N contains a 21-amino-acid linker, while GZF3-N and nGZF3-N contain a short AAARA linker between the zinc-finger modules and the Fok1-N domain. “P” stands for promoter CMV, PGK, or HSV-tk. (B) Western blot. Transfected 293T cells were harvested after 24 and 48 h and cell lysates probed with an anti-HA antibody. “cto” indicates transfection with empty vector. (C) Apoptosis. The sub-G1 population of transfected 293T cells was determined by flow cytometry of PI-stained cells after 48 h and is shown as the fraction of transfected cells. **Statistically significant (P < 0.05).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Episomal gene repair assay. (A) Experimental setup. Repair and target plasmids are described in the text. The stop codons (bold, italics) and the recognition site for I-SceI (boxed) are highlighted. (B, C) Episomal gene repair. 293T cells transfected with repair plasmid (RP), target plasmid, and a nuclease expression vector were analyzed by flow cytometry after 2 days. The graph displays the fraction of EGFP-positive cells in relation to transfected cells. The presence or absence of RP is indicated by + or −, “cto” indicates control. In (B), cells were transfected with target plasmid “3-3” along with plasmids encoding different promoters and types of GZF3 nucleases. **Statistically significant (P < 0.01) stimulation of SDR; “nd” stands for “not determined” in the case in which the number of REx-positive cells was below 1% due to cell death. In (C), cells were transfected with “0-0”, “1-1”, “2-2”, “3-3”, or “4-4”, along with nuclease expression plasmids. **Statistically significant (P < 0.01) stimulation of HR comparing gene repair in the absence (“0-0”) and in the presence of respective binding sites (e.g., “1-1”).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
SDR stimulated by heterodimeric nucleases. (A) Episomal gene repair. 293T cells were transfected with “3-1” and plasmids coding for I-SceI, GZF1-N, and GZF3-N as indicated by +. **Statistically significant stimulation of gene repair (P < 0.001). (B) Chromosomal gene repair. 293/3-1 cells, which contain a single copy of target locus “3-1”, were transfected with repair plasmid (RP) and endonucleases as indicated by + (0.25 μg) and ‡ (0.75 μg). The columns designate the fraction of EGFP-positive cells 7 days posttransfection normalized for transfection efficiency. The presence or absence of RP is indicated by + or −. **Statistically significant stimulation (P < 0.01) of gene repair above basal HR. (C) Cytotoxicity. 293/3-1 cells were transfected with the same amounts of endonuclease expression vectors as for (B). The percentage of dead 293/3-1 cells was determined after 48 h by analyzing PI incorporation. The number of PI-positive cells is shown as a fraction of transfected cells.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions