GT 39) primers. These primers introduced a TA at the 59-end of each construct except the CRX mutant and wild-type constructs (see below). The PCR reactions were performed using murine and human genomic DNA as the template and were subjected to a hot start of 5 min at 94uC prior to the addition of the Platinium Taq DNA Polymerase High Fidelity (Invitrogen) followed by 7 cycles amplification: denaturing for 25 sec at 94uC, annealing for 30 sec to 7 min at 66uC (depending on the size of the PCR product), 35 cycles amplification: denaturing for 25 sec at 94uC, annealing for 30 sec to 7 min at 60uC (depending on the size of the PCR product) and a final extension at 66uC for 10 min. The primers used for each construct were listed in Table S1. The PCR products were purified after gel agarose electrophoresis using the gel extraction kit (Qiagen). The BP reaction was performed using 300 ng of the PCR product, 300 ng of the pDONR vector (Invitrogen), 4 ml of the BP Clonase Reaction Buffer (Invitrogen) and 4 ml BP Clonase enzyme mix (Invitrogen) to obtain entry clones. The reaction was incubated one hour at room tempera- ture. OneShot TOP10 chemically competent E.coli cells (Invitro- gen) were transformed by using 5 ml of the BP reaction. Positive clones were identified by restriction enzyme analysis of purified plasmid and verified by DNA sequencing. The LR reaction was performed by using 300 ng of the entry clone, 300 ng of the destination vector (pGL4.17 or peGFP), 4 ml of LR Clonase Reaction Buffer (Invitrogen) and 4 ml of the LR Clonase enzyme (Invitrogen) to obtain the expression vectors. The reaction was incubated one hour at room temperature. OneShot TOP10 chemically competent E.coli cells (Invitrogen) were transformed by using 5 ml of the LR reaction. Positive clones were identified by restriction enzyme analysis. Construction of mutated CRX binding site Plasmids containing a mutated CRX binding site (the TAA in the TAAT core recognition site was modified to CCG) were generated by DNA synthesis (DNA2.0) and cloned in pDONR. The LR reaction was performed as previously described to subclone the sequences into pGL4.17 and peGFP. For the synthesis, the consensus sequence from GenBank (NCBI) was used. Because this sequence contained some sequence variants from the above described PCR generated constructs (due to both sequence variations in the donor DNA sample and to nucleotide differences in the PCR primer sequences) and because for the CRX mutation analysis we wanted the mutant constructs to differ from the wild-type constructs only in at the CRX binding site, we used the synthesized constructs as templates to generate wild-type constructs that were indeed identical except at the CRX site. A ‘mutagenic’ primer was used to convert the CCG sequence back to the wild type CRX binding site (TAA) by PCR-based mutagenesis. The resulting fragment was then used as a mega-primer to amplify the full-length construct in a second round of PCR amplification. The full-length wild-type constructs were then cloned back into pDONR on an AvaI –EcoRV fragment, and the resulting plasmids were sequenced. Reporter constructs were generated by Gateway recombination as described above. RNA extraction and RT-PCR Total RNA was extracted from Y79 cells using the RNeasy extraction kit (Qiagen). One microgram of total RNA was reverse transcribed into cDNA by incubation with 1 ml (500 mM final) of deoxyribonucleoside triphosphate solution (dNTP), 1 ml of oligodT (10 mM), 4 ml 56 reaction buffer, 2 ml of 0.1 M dithiotreitol (DTT), 1 ml of RNasin an 200 U of superscript II (Invitrogen) to a final volume of 20 ml. Samples were incubated 1 hour at 37uC and the enzyme was inactivated at 72uC during 10 min. The resulting CRX Regulates RdCVF cDNA were amplified in PCRs using Platinium Taq High Fidelity (Invitrogen) in 2 mM MgSO4, 200 mM dNTP and 50 pmol of primers. Oligonucleotides used were: human CRX forward: 59 GGC TCT GAA GAT CAA TCT GCC TGA 39, reverse: 59 CCA TAG CTC TGG CCT GAT AGG GAG 39: human G6PDH forward: 59 ATG TTCGTC ATG GGT GTG AAC C 39, reverse: 59 AGG GAT GAT GTT CTT CTG GAG AGC C 39. Cycling protocol was: 94uC, 2 min; 35 cycles of 94uC, 30 sec; 60uC, 30 sec; 72uC, 2 min, followed by a final extension step at 72uC for 5 min. PCR products were run on a 1.5% agarose gel stained with ethidium bromide and visualized under UV illumination. Transient transfection and reporter gene assay Luciferase activity was determined using a luciferase assay system (Promega) according to the manufacturer’s protocol. Briefly, Y79 and HEK 293 cells were plated for 24 h at respectively 56105 and 26105 cells/well in 24-well plates. Five hundred (for Y79) or 100 ng (for HEK 293) of promoter constructs or the empty vector (pGL4.17) was transiently transfected into the cells with Lipofectamine 2000 (Invitrogen). Cells were co- transfected with pRL-TK containing Renilla luciferase gene driven by the thymidine kinase promoter (Promega). One day after transfection, cell extract was lysed, and the luciferase activity of the extract was measured with the luminometer LB-96V (Berthold). Firefly luciferase activities were normalized by Renilla luciferase activities and relative luciferase activities were expressed as fold induction as compared to the empty vector (pGL4.17). Electroporation of retinal explants Mouse retinas from newborns were electroporated the day of birth as described in Matsuda & Cepko [33,40]. Briefly, newborn retinas were dissected in Hank’s Balance Sodium Salts (HBSS) without calcium and magnesium (Invitrogen) and were cut into 3 pieces and were submerged in a 1 mg/ml DNA solution. They were subjected to 5 electric pulses at 25 V with 50 ms duration and 500 ms intervals using a BTX ECM830 square wave generator (BTX). Explants were then placed on polycarbonate filter discs (Dutscher) and cultured in DMEM/F12 medium (Invitrogen) with 10 mM HEPES pH 7.0 containing 5% FBS (Invitrogen). Cultures were maintained up to 9 days and media were changed every 2–3 days. The empty vector pCIG-eGFP used as control expresses the eGFP under the control of the chicken b- actin promoter [40]. Image acquisition The fluorescence of retinal explants was monitored after electroporation. Fluorescent signals were captured with a macro- scope Z6 APO (Leica) equipped with a CCD CoolSnap camera (Roper Scientific). MetaMorph software (Universal Imaging Corporation) was used to quantify the intensity of the emitted fluorescence Immunofluorescence Retinal explants were fixed with 4% paraformaldehyde (PAF) in PBS for 10 min and incubated 30 min in 30% sucrose at room temperature. Retinal explants were frozen in PBS-10% sucrose- 7.5% gelatin using a immersion in dry ice and isopentane. The samples were cut into 10 mm thick sections and mounted on glass slides. To reduce non-specific labeling, the sections were incubated for 2 h at room temperature in blocking solution (PBS, 0.1% Triton X-100 and 2% goat serum). The sections were incubated with the primary GFP- (Roche) and RFP- (Mbl) antibodies in PLoS ONE | www.plosone.org 14 October 2010 | Volume 5 | Issue 10 | e13075