once a week for each treated chicken using previously published methods [15]. Two high contrast, vertical square wave grating stimuli with spatial frequencies of 0.065 or 0.26 cycles?degree21 (bar widths of 5 cm and 1.25 cm, respectively) were used to elicit the responses. The behaviors were recorded using a digital camera (Canon Vixia HF20 HD video camera; http://www.usa.canon. com). The recorded behaviors were analyzed using a zero-three point scoring system: zero, no OKN response; one, inconsistent or unidirectional responses to the lower spatial frequency grating; two, consistent bidirectional responses to the lower spatial frequency grating; three, consistent bidirectional responses to the higher spatial frequency grating. The scores of the OKN responses for the right and left eyes of each animal obtained using the 5 or 1.25 cm bar stimuli were averaged to obtain the OKN response score for each stimulus on each test day. Retinal Whole-Mounts and Immunohistochemistry Retinal whole mounts and immunohistochemistry were carried out as previously described [20]. The GFP and mCher reporter proteins were detected using either native fluorescence or immunohistochemistry. GFP and mCher native fluorescence was detected in retinal whole mounts and sections using the appropriate fluorescence filters and documented using a Zeiss AxioCam MRm digital camera. For immunohistochemistry, retinal whole mounts or cryosections (10 mm) were probed with a polyclonal rabbit anti-GFP antibody (1:2000, overnight, kindly provided by W. Clay Smith, University of Florida, Gainesville, FL) and counterstained with 49,6-diamidino-2-phenylindole (DAPI) where indicated. The primary antibody was detected using Alexa Fluor 488 (1:500, Invitrogen). Zeiss filter set 02 was used to visualize DAPI-stained cell nuclei. To visualize GFP expression in retinal whole-mounts, retinas were incubated with a biotinylated, anti-rabbit secondary antibody and were processed using a Vectastain ABC kit and a 3,39-diaminobenzidine (DAB) substrate kit for peroxidase (Vector laboratories, Inc., Burlingame, CA). The percent transduction of the retinal whole mounts was determined using the analyze particles feature of ImageJ version 1.44 h. Western Blot Western blot analyses of the proteins expressed in transfected or transduced HEK 293FT cells and in transduced retinas were performed as previously described [47]. In brief, protein samples (1 mg of total transfected cell lysate, 15 mg of total transduced cell lysate, or 30 mg retina lysate) were separated on precast, gradient 4–12% polyacrylamide gels and transferred to PVDF membranes using an i-Blot transfer apparatus according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The blots were prepared in quadruplicate permitting each blot to be probed with a single antibody. Antibodies recognizing GFP, 2A, hemagglutinin, GC1, or GAPDH were used to probe the blots. After blocking the membranes in 5% milk/Tris Buffered Saline (TBS) for 1 h, the blots were incubated with primary antibody overnight at 4uC with gentle shaking. After three 10 minute washes in TBS, blots were incubated in secondary antibody diluted 1:5000 in 5% milk/TBS for two hours at room temperature. The secondary antibodies used in this study were anti-chicken (GeneTex, Irvine, CA), anti- rabbit and anti-mouse (Cell Signaling Technology, Inc., Danvers, MA), and anti-rat (Sigma-Aldrich, St. Louis, MO). The blots were then washed and signals were visualized with ECL (Amersham, UK). Films were scanned using a GS-800 calibrated densitometer (BioRad, Hercules, CA) and the signals were quantified using Scion Image software (Scion Corp., Frederick, MD). For each bicistronic transgene, the densities of the mCherH and GFP-2A protein bands detected in the samples were determined and then Lentiviral Delivery of Proteins Using 2A Peptides normalized to the highest mCherH or GFP-2A sample value, respectively, which was assigned a value of 1.0. The relative levels of GFP-2A and mCherH produced by the transgenes were compared using analyses of variance and Tukey post hoc tests when appropriate (SigmaStat v2.03, Aspire Software Internation- al, Ashburn, VA). Graphs were generated using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA). Primary Antibodies The primary antibodies used in the western blot experiments were chicken anti-GFP (1:3000, Abcam, Cambridge, MA, Western blot analysis), rabbit anti-2A (1:2000, Millipore, Billerica, MA), rabbit anti-GC1 (1:500, kind gift from Dr. A. Yamazaki), rat anti-hemagglutinin, High Affinity (1:5000, Roche, Indianapolis, IN), and mouse anti-GAPDH (1:5000, Encor, Gainesville, FL). Rabbit anti-GFP (1:2000, kind gift from Dr. W. Clay Smith) was used to stain retinal whole mounts. Supporting Information Figure S1 Cleavage of GFP-2A-mCher and GFP-furin-2A- mCher polypeptides in transfected HEK 293FT cells. (A, B) Diagrams of the EF1-GFP-2A-mCher and EF1-GFP-furin (F)-2A- mCher transgenes. (C) Western blot showing cleavage of EF1- GFP-2A-mCher (-F) and EF1-GFP-F-2A-mCher (+F) transgenes. The blot was probed with an antibody against GFP that recognized both the cleaved and uncleaved polypeptides. The amount of total protein loaded per lane was either 20 or 50 mg. (TIF) Video S1 Optokinetic behavior of 8-day-old wild-type chicken. The responses shown were elicited by a high-contrast vertical square wave grating with a spatial frequency of 0.26 cycles?de- gree21 (bar width 1.25 cm). Reflexive head movements charac- teristic of the optokinetic response in birds were observed when the stimulus was rotated either clockwise or counter clockwise. No optokinetic responses were elicited by the white control stimulus (MP4) Video S2 Optokinetic behavior of 8-day-old untreated GU- CY1*B chicken. No responses were elicited by either of the high- contrast vertical square wave grating stimuli. The stimuli used in this particular test were a high-contrast vertical square wave grating with a spatial frequency of 0.26 cycles?degree21 (bar width 1.25 cm) and a solid white stimulus. (MP4) Video S3 Optokinetic behavior of 8-day-old GUCY1*B chicken that had been treated with pFIN-EF1-GFP-2A-GC1-WPRE lentivirus on embryonic day 2. This treated animal, unlike untreated animals, exhibited a robust, optokinetic response that was assigned a score of 3.0. Reflex responses were elicited by the high-contrast vertical square wave grating (bar width 1.25 cm) when rotated in either clockwise or counter clockwise. No responses were elicited by the solid white control stimulus. (MP4) Acknowledgments The authors would like to acknowledge Dr. W. Clay Smith for providing the rabbit polyclonal GFP antibody and Dr. Akio Yamazaki for providing the rabbit polyclonal GC-1antibody. Author Contributions Conceived and designed the experiments: JDV WEC MG MH EW DD IM KSE SSR. Performed the experiments: JDV WEC MG MH EW DD PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e20553