2A vectors that will permit us to determine if combining GC1 delivery with delivery of additional therapeutic proteins can permanently restore vision to these animals. While we use lentivectors in our studies, 2A-like cleavage peptides should prove valuable in any therapeutic or biotechnological application requiring co-expression of multiple proteins in targeted cells. Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal protocols were approved by the University of Florida Institutional Animal Care and Use Committee (Approval 201004563) and adhered to the policies outlined in the Guide for the Care and Use of Laboratory Animals. Construction of Vectors pFIN-EF1-GFP-EF1-mCherH-WPRE. pFIN-WPRE [16] was digested with NheI. GFP was amplified using PCR and a sense primer containing NheI and BsiWI and an antisense primer containing MluI and NheI. The PCR product was ligated into pFIN-WPRE at the NheI site to create pFIN-GFP-WPRE. This vector was then linearized using NotI and the EF1 promoter, which was amplified using sense and antisense primers containing NotI, was ligated into pFIN-GFP-WPRE to create pFIN-EF1- GFP-WPRE. A second EF1 promoter was amplified using a sense primer containing BsiWI and an antisense primer containing PacI and MluI sites and was ligated into pFIN-EF1-GFP-WPRE using BsiWI and MluI. Finally, hemagglutinin (HA)-tagged mCherry (mCherH) was created using PCR primers containing PacI sites and was ligated into the linearized pFIN-EF1a-GFP- EF1-WPRE to create the final construct. The antisense primer included the HA tag that was fused in frame to mCher immediately upstream of the stop codon. It was necessary to tag mCher with an epitope to which antibodies exist since we and others [45] have found that the antibodies that are currently available for mCher fail to detect this protein on western blots. pFIN-EF1-GFP-WPRE-EF1-mCherH-WPRE. pFIN-EF1-GFP- EF1-mCherH-WPRE was linearized using BsiWI. WPRE was amplified using sense and antisense primers containing BsiWI sites and WPRE was ligated into the linearized backbone to create pFIN-EF1-GFP-WPRE-EF1-mCherH-WPRE. pFIN-EF1-GFP-2A-mCherH-WPRE. pFIN-EF1-GFP-WPRE [16] was digested with NheI to remove GFP. GFP-2A-mCherH was created using a three-step PCR strategy described by Symczak et al [46] that included the addition of a GSG linker sequence upstream of the PTV1 2A peptide sequence. The final set of primers used to amplify GFP-2A-mCherH contained NheI sites that permitted it to be ligated into the NheI site in the pFIN- EF1a-WPRE backbone to create the final vector. pFIN-EF1-GFP-Furin-2A-mCher-WPRE. This vector was created using the same strategy as that used to construct pFIN- EF1-GFP-2A-mCherH-WPRE except that the primers used to amplify GFP included sequence encoding the furin cleavage site (RAKR) that was inserted in frame just upstream of the GSG linker. pFIN-EF1-GFP-2A-GC1-WPRE. The cDNA encoding bovine GC1 was removed from pBSII SK+-GC1 [15] using SpeI and KpnI and was ligated into our pFIN-WPRE backbone that had been digested with SpeI and KpnI. EF1 was amplified using sense and antisense primers containing NotI sites and was ligated into the NotI site in pFIN-GC1-WPRE. The resulting Lentiviral Delivery of Proteins Using 2A Peptides vector, pFIN-EF1-GC1-WPRE, was then digested with NheI and StuI, the StuI site being located approximately 700bp downstream from the start codon of the GC1 open reading frame. GFP-2A- GC1 (5’ 700 bp) was amplified using the three-step PCR method. The final sense and antisense primers used to amplify the GFP-2A- GC1 (5’ 700 bp) fragment contained NheI and StuI sites, respectively. GFP-2A-GC1 was then ligated into pFIN-EF1- GC1-WPRE to create pFIN-EF1-GFP-2A-GC1-WPRE. Lentiviral Packaging The lentiviral vectors were packaged into lentivirus pseudotyped using vesicular stomatitis virus G (VSV-G) glycoprotein using a three plasmid packaging system as previously described [20]. Viral titers were estimated using a Lenti-X qRT-PCR kit (Chemicon, Billerica, MA) and typically averaged 261012 viral genomes per ml. Cell Culture and In Vitro Transfection and Transduction HEK 293FT cells were grown and maintained in Dulbecco’s Modified Eagle high glucose media containing 4.5 mg/ml D- glucose, L-glutamine and 0.11 mg/ml sodium pyruvate (Invitro- gen, Carlsbad, CA) to which was added 10% fetal calf serum, 50 U/ml penicillin G, 50 mg/ml streptomycin, and 500 mg/ml geneticin. The cells were transiently transfected with plasmid DNA using Superfect (Qiagen, Valencia, CA) according to the manufacturer’s instructions. In brief, 200,000 cells were seeded per well of gelatin-coated 6 well plates 24 h prior to transfection and were transfected with a mixture of 2.0 mg of DNA and 10 ml of Superfect per well. The transfected cells were allowed to proliferate for 48 h. To analyze the performance of the vectors when delivered as viral particles, HEK 293FT cells were plated as described above and were exposed to approximately 26109 viral particles for 72 h prior to analysis. Transfected or transduced cells were either harvested for Western blot analyses in SDS sample buffer supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN) or were examined directly using a Zeiss AxioCam MRm digital camera system (Carl Zeiss Microimaging, Inc.,Thornwood, NY). GFP was detected using a narrow-band GFP filter set 41020 (Chroma Technology Corp, Bellows Falls, VT) and mCher was detected using a Chroma custom filter set that consisted of an exciter ET572/35, an emitter ET632/60, and a beamsplitter (Chroma Technology Corp, Bellows Falls, VT). For comparison purposes the exposure times used to photograph each reporter were kept constant between constructs. Hoechst (Molec- ular Probes Inc., Eugene, OR) was added to live cells for 3 min before analysis to visualize nuclei. Viral Injections of Chicken Embryos All eggs used in this study were obtained from our wild-type Rhode Island Red and our GUCY1*B breeding colonies that we maintain at the University of Florida. Lentiviruses were injected into chicken embryos (stage 8–10) on embryonic day 2 (E2) as previously described [15]. Approximately 345 nl of virus was injected into the anterior region of the developing embryonic neural tube. The treated eggs were then sealed and incubated to E20 at which time the embryos were either sacrificed and the retinas were harvested for analyses or were hatched using our previously described protocol [15]. Behavioral Analyses The optokinetic nystagmus (OKN) responses, which are reflexive visual responses driven primarily by visual stimuli processed by the peripheral regions of the retina, were evaluated PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e20553