pyramidal well blocks in a Microtitertron shaker (Appropriate Technical Resources, Inc.) at 350 rpm, 37uC, 80% humidity. These cultures were grown in NM medium supplemented with 30 mg/ml chloramphenicol, 30 mg/ml kanamycin, and 50 mM IPTG. Following overnight growth, selection strain cultures were infected with a matched combinatorial zinc finger array phagemid phage library constructed as described above. Following phage infection, 4 ml of NM medium [46] supplemented with 30 mg/ml chloramphenicol, 30 mg/ml kanamycin, and 50 mM IPTG was added to the cells which were then shaken on the Microtitertron shaker for 1.5 hrs. The infected cells were then spun down and 4ml of the supernatant removed. The cell pellet was then resuspended in the remaining 1 ml of liquid media and 250 ml of this resuspension was plated on two different NM/CCK medium plates containing 50 mM IPTG, 10 mM 3AT, and 20 mg/mL streptomycin or 50 mM IPTG, 25 mM 3AT, and 40 mg/mL streptomycin. After 36–48 hours of incubation, colonies were harvested from the highest stringency plate yielding at least 1000 colonies as previously described [26]. The resulting cell suspension was then diluted with 4.5 ml 2XYT media supplemented with 50 mg/ml carbenicillin and 30 mg/ml kana- mycin to an OD600<0.1 in the 10 ml-capacity well of a 24-well block and allowed to grow for 1 hour in the Microtitertron shaker as described above. This subculture was infected with 1011 kanamycin transducing units of M13K07 helper phage and then grown for six hours in the Microtitertron shaker. Phage-containing culture supernatants were harvested by filtering the cell cultures through a 0.22 mm polyethersulfone syringe filter. In the second stage of OPEN selection, selection strain cells were again grown in 24-well blocks but in 1 ml of NM medium supplemented with 30 mg/ml chloramphenicol, 30 mg/ml kana- mycin, and no IPTG. This overnight culture was infected with ,66105 ampicillin-transducing units (ATUs) of zinc finger- encoding phagemid phage rescued from the initial stage of selection. Following infection, 400 ml of NM medium supplement- ed with 30 mg/ml chloramphenicol and 30 mg/ml kanamycin was added to the cells which were then shaken on the Microtitertron shaker for 1.5 hrs. 375 ml of this infected culture (corresponding to ,561025 infected/transformed cells) was then plated on a square 100mm6100mm NM medium agar plate supplemented with 100 mg/ml carbenicillin, 30 mg/ml chloramphenicol, 30 mg/ml kanamycin and containing gradients of 3-aminotriazole (from 0 to 80 mM) and streptomycin (from 0 to 100 mg/ml). Gradient plates were poured as previously described [26]. Construction of ZFN expression vectors DNA sequences encoding zinc finger arrays identified by OPEN were transferred to ZFN expression vectors by using the phagemids encoding these arrays as templates for PCR reactions using primers OK.1677 and OK.1678 (Supplemental Ta- ble 26). The resulting DNA fragments (encoding the zinc finger arrays) were digested with XbaI and BamHI and cloned into XbaI/ BamHI-digested ZFN expression vectors pMLM335 or pMLM336 [26]. The pMLM335 and pMLM336 vectors encode previously described obligate heterodimeric ZFNs [43]. Final sequence- verified plasmids were prepared using a QIAgen HiSpeed Midiprep kit using RNase free reagents and stored in RNase- free Eppendorf Safe Lock Tubes. Preparation of ZFN-encoding RNA ZFN expression vectors were linearized with PmeI (an enzyme which cleaves just 39 to the end of the ZFN coding sequence) and transcribed in vitro using the T7 mMessage mMachine kit Rapid Mutation by OPEN ZFNs (Ambion). The transcribed ZFN RNAs were then polyadenylated using the Poly(A) Tailing kit (Ambion). Injection of zebrafish and analysis of somatic mutations Approximately 2 nl of the ZFN RNA (at concentrations of 50– 400 pg/nl) was injected into one-cell stage zebrafish embryos. Two days following fertilization, the surviving injected embryos were grouped into either ‘‘normal’’ or ‘‘deformed’’ phenotypes. Genomic DNA was extracted from pools of 4–12 embryos from each ‘‘normal’’ group using DNA extraction buffer (10 mM Tris, pH 8.0, 200 mM NaCl, 10 mM EDTA, 0.5% SDS, 100 mg/ml Proteinase K), followed by phenol/chloroform extraction and ethanol precipitation. The DNA was resuspended in 40 ml of TE (10 mM Tris, pH 8.0, 1 mM EDTA). 2.5 ml of the resulting genomic DNA was then used as template for a PCR reaction using Platinum Taq DNA Polymerase High Fidelity enzyme (Invitrogen) with primers designed to anneal approximately 150 to 200 bp upstream and downstream from the expected mutation. The resulting PCR product was cleaned up using a QIAGEN Minelute PCR purification kit and then ligated using a ZeroBlunt TOPO kit (Invitrogen) into linearized pCR4 Blunt-TOPO vector. The ligation was transformed into Mach1 T1-bacteriophage resistant E.coli (Invitrogen) and plated on LB plates containing 50 mg/ml kanamycin. Following incubation overnight at 37uC, colonies were picked from these plates and inoculated into 700 ml TB medium containing 50 mg/ml kana- mycin in 96-well blocks with 1ml pyramidal-bottom wells. These blocks were shaken at 900 rpm, 37uC, and 80% humidity in a Microtitertron shaker. Plasmid DNA was isolated from these cultures and sent for sequencing using the ‘‘T3 sequencing’’ primer (Supplemental Table 26). Identification and sequencing of germline transmitted mutations Potential founders were crossed with wild-type zebrafish. One to three dpf (days post fertilization), progeny were lysed individually in lysis buffer (10 mM Tris, pH 8.0, 2 mM EDTA, 0.1% Triton X-100, 100 mg/ml Proteinase K) and incubated at 50uC overnight. For each target gene, 10–12 embryos from each potential founder were screened for the presence of ZFN-induced mutations by amplifying the region surrounding the relevant ZFN cleavage site by PCR and then using either restriction digest- and/or DNA sequencing-based assays: For the tfr2 gene, we used primers OK.1922 and OK.1923 to amplify the region surrounding the ZFN target site by PCR and the resulting ,405 bp product from each embryo was directly sequenced with primer Tfr2-seq (Supplemental Table 26). For the dopamine transporter gene, we used primers OK.1916 and OK.1917 (Supple- mental Table 26) to amplify the region surrounding the ZFN target site by PCR and the resulting ,418 bp product was digested with the restriction enzyme ApeKI. The PCR product from a wild- type allele will yield 5 fragments of 251, 68, 52, 25 and 7-bp sizes. Introduction of indel mutations at the ZFN target site will cause disruption of the ApeKI site and result in the appearance of an additional 120-bp fragment which is detectable on a 3% agarose gel. PCR fragments from selected progeny that showed evidence for loss of the ApeKI site were blunt-end cloned into the pCR4 Blunt-TOPO vector as described above and sequenced with the ‘‘T3 sequencing’’ primer. For the telomerase gene, we used primers OK.1928 and OK.1930 (Supplemental Table 26) to amplify the surrounding region by PCR and the resulting ,306 bp product was digested with the enzyme BspHI. The PCR product from a wild-type allele will contain only one BspHI site. Introduction of indel mutations at the ZFN target site will disrupt the BspHI site thereby resulting in the generation of PCR products resistant to digestion by BspHI. PCR PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4348