PMC:2669177 / 32177-41626 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/2669177","sourcedb":"PMC","sourceid":"2669177","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2669177","text":"Materials and Methods\n\nEthics statement\nAll animal procedures were approved by the University of Oklahoma Health Science Center Institutional Animal Care and Use Committee (IACUC) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (see Policies, http://www.arvo.org/).\n\nVector and nanoparticle construction\nTwo constructs were generated expressing the full-length mouse Rds cDNA (1.7 kb) containing the P341Q modification (called NMP), for specific detection with the mAB 3B6 monoclonal antibody [18]. Either the human interphotoreceptor retinoid-binding protein (IRBP) promoter (1.3 kb) [33] or the chicken beta-actin (CBA) promoter (280 bp) was used to drive NMP expression. The two promoter regions were amplified from genomic DNA by PCR, sequenced, and then sub-cloned into the pXL-TOPO vector in front of NMP using EcoR I and BamH I restriction enzymes. The two plasmid DNAs were individually compacted into rod-like acetate nanoparticles (Figure S1) at Copernicus Therapeutics as reported previously [12], [13], and were used at a final concentration of 3.06 µg/µl in 0.9% saline.\n\nSubretinal injections\nMice (rds +/− pups at P5) were anesthetized by incubation on ice for 2–2.5 minutes. The eyelid of the right eye was cut, the cornea was exposed, and a puncture in the cornea was made with a sterile 30-gauge needle. A 35-gauge blunt-end needle attached to a 10 µl Nanofil® syringe (World Precision Instruments, Sarasota FL) was inserted into the puncture under an operating microscope (Carl Zeiss Surgical, Inc., NY). A volume (0.3 µl) of solution containing fluorescein dye and either nanoparticles, saline (vehicle), or naked DNA was delivered into the subretinal space, usually in the superior temporal quadrant. Since the retina is not fully developed at this age, some injected material is likely released into the vitreous, although the site of injection is subretinal. After injection, the needle was left in place for 3–5 seconds to allow full treatment delivery before being withdrawn gently. Successful delivery of material was confirmed by observation of subretinal yellow-green fluorescence at the time of injection. The cut eyelid was returned to its original position and the surface of the eye was gently blotted with a Kimwipe. Animals were warmed on a temperature-controlled (37°C) bed until fully awake. All nanoparticles and uncompacted plasmid DNA (naked DNA) were used at the same concentration (3.06 µg/µl), selected based on data from our previous study [13]. Because the compaction process relies on the presence of DNA, there is no “empty” nanoparticle, so controls were limited to saline and naked DNA carrying the same therapeutic vector as the nanoparticles. If material delivery could not be confirmed, or if microphthalmia or signs of intraocular infection were observed, the injection was considered unsuccessful and the animal was removed from the study (121/432∼28%). Mice were maintained in the breeding colony under cyclic light (14-hour light/10-hour dark) conditions; cage illumination was approximately 7 foot-candles during the light cycle.\n\nRNA isolation and qRT-PCR\nBoth injected and uninjected whole eyes were collected at PI-2, 7, 14, 21, 30, and 120 days for analysis of mRNA levels. qRT-PCR was performed with a MyIQ single-color qRT-PCR machine (Bio-Rad). Mice were euthanized, eyes were enucleated and homogenized and total RNA was extracted using TRIzol (Invitrogen Inc. Carlsbad, CA) as described previously [13]. Subsequently, DNase treatment was performed with RNase-free DNase (Promega Inc.) to remove both genomic DNA and any remaining nanoparticle DNA. cDNA synthesis by reverse transcription was performed and 20 ng of cDNA from each sample was used for qPCR. Rds primer sequences were reported previously [13]. Melting curve analysis and agarose gel electrophoresis were performed at the end of the reaction to ensure that the PCR products were specific and of appropriate size. All experimental mRNA levels were quantified against the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) as described previously [13]. Relative expression levels were calculated by the 2−ΔcT method [48]. At least three injected and three uninjected eyes from each treatment group at each of the scheduled time points were analyzed. Two independent qPCR experiments for each set of samples were performed and individual samples were run in triplicate. For each sample, the six values (three from two separate reactions) were averaged to get an expression value. The S.E.M. shown in Figure 1 represents the variation from sample to sample (i.e. inter-mouse variation). Rds primers amplify from transferred Rds and WT Rds, but not from the Rds mutant allele. To confirm that Rds levels were not artificially altered by the presence of undigested nanoparticle, control reactions amplifying from the IRBP or CBA promoter regions were performed and no product was detected. As an additional control, a subset of samples was analyzed with Rds primers, but without the addition of reverse transcriptase and no amplification was detected.\n\nAntibodies\nAntibodies were procured and used for immunohistochemistry (IHC) and Western blot (WB) analysis as follows: mAB 3B6 (a kind gift from Dr. R.S. Molday, University of British Columbia, Vancouver, BC, Canada, IHC-1∶100); RDS-CT recognizing both endogenous Rds and, to a lesser extent, NMP (generated in-house, IHC-1∶100, WB-1∶1000); anti-S-opsin, recognizing short-wavelength mouse cone opsin (generated in-house, IHC-1∶100, WB-1∶1000); mAb 1D4, recognizing rhodopsin (a generous gift from Dr. R.S. Molday, WB-1∶5000); Rom-1 (generated in-house, WB-1∶1000); and β-actin-HRP (Sigma/Aldrich, WB-1∶5000).\n\nImmunohistochemistry\nWhole eyes were enucleated and fixed with phosphate-buffered saline containing 4% paraformaldehyde at 4°C overnight. With the exception of PI-2 eyes, the cornea and lens were removed and the eye was returned to fixative for an additional two hours. The eyes were cryoprotected by serial immersion in 15% and 30% (w/v) sucrose solutions for at least two hours each. Individual eyes were embedded in M1 embedding medium (Thermo Electron Corporation, PA) and frozen on dry ice; frozen sections (10 µm thickness) aligned with the vertical meridian were cut with a cryostat (Leica) and collected on precleaned Superfrost-plus® microscope slides (Fisher Scientific). The entire eye was sectioned, and every sixth section was collected, enabling examination of gene expression throughout the retina. For immunohistochemistry, all steps were carried out at room temperature as described previously [13], [47]. Staining controls included eyes from age-matched NMP transgenic and WT mice, and slides on which primary or secondary antibodies were omitted. Observation and imaging were performed using an epifluorescent microscope (AxiophotZeiss Ltd., Germany) and a spinning disk confocal microscope (BX62 Olympus, Japan).\n\nProtein detection by western blot\nWestern analysis was performed as reported previously [24], [35], [49]. Dissected individual retinas were homogenized on ice and solubilized (50 mM Tris, pH 7.8, 100 mM NaCl, 5 mM EDTA, 0.05% SDS, 1 mM PMSF, 1% TX-100, 2.5% (v/v) glycerol) for one hour at 4°C, then processed for SDS-PAGE and subsequent Western blotting using 10–50 µg protein per lane (as detected by Bradford assay; Bio-Rad). Blots were imaged using a Kodak Image Station 4000R with Kodak MI software.\n\nElectroretinography\nFull-field electroretinography was performed as previously reported [40] and analysis of the obtained electroretinograms (ERGs) was performed as described in detail elsewhere [24], [40] to quantitatively assess rod- and cone-mediated visual function. Both scotopic (rod) and photopic (cone) ERGs were recorded at PI-30, PI-60, and PI-120 on animals from all treatment groups and controls.\n\nHistology and electron microscopy\nEnucleated eyes were fixed, sectioned, and processed as described previously [50]. Semithin sections collected through the vertical meridian were stained with 1% (w/v) toluidine blue in 1% (w/v) sodium borate, coverslipped, and viewed with an Olympus BH-2 microscope under 63×. Digital images were captured using a Nikon DXM1200 digital camera. Ultrathin sections were stained with 2% uranyl acetate and lead citrate and imaged using a JEOL 100CX electron microscope (80 KeV).\n\nStatistical analyses\nFor qRT-PCR data, values are expressed as ratios (to uninjected eye) of mean relative expression (±S.E.M.). Since ratios have an inherently skewed (non-Gaussian) distribution, data were log-transformed before undergoing one-way ANOVA. For ERG data, nanoparticle-injected groups were compared with naked DNA-injected groups. All groups passed the Kolmogorov-Smirnov test for normality and P-values are from two-tailed, unpaired Student's t-tests. In cases where unequal variance was found (per an F-test), Welch's correction was applied (http://web.uccs.edu/lbecker/SPSS/ttest.htm). Means and standard errors are reported in Table 1 as indicated. To determine whether there was any significant change in ERG amplitude over time, two-way ANOVA with Bonferroni's post-hoc test was used to compare treatment groups at different ages. To determine whether naked DNA caused any effect on function, one-way ANOVA with Bonferroni's post-hoc test was used to compare CBA-NMP naked DNA, IRBP-NMP naked DNA, and saline injected 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