Animal and Human Tissues
C57Bl/6J and rd mice were obtained from our colonies, bred from stock originated at the Jackson Laboratories (Bar Harbor, ME). Mouse eyes were quickly enucleated after death and the retinas dissected and frozen. All experiments were conducted in accordance with the approved UCLA Animal Care and Use Committee protocol and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Retinas from 2-year-old normal and cd dogs were kindly provided by Gustavo Aguirre (University of Pennsylvania, School of Veterinary Medicine). Healthy human donor eyes were obtained from the National Disease Research Interchange (Philadelphia, PA) and immediately frozen in liquid nitrogen. The donor eyes were managed in compliance with the Declaration of Helsinki.
Total RNA was extracted from mouse and human retinas (TRIzol; Invitrogen, Carlsbad, CA). Poly A+ RNA was obtained with an mRNA purification kit (Oligotex; Qiagen, Valencia, CA). RNA quality was assessed with a bioanalyzer (model 2100; Agilent Technologies, Palo Alto, CA), quantified (NanoDrop spectrophotometer; NanoDrop Technologies, Wilmington, DE), and stored at −80°C.
Representational difference analysis (RDA) subtraction of mRNAs from adult normal and cd
dog retinas was performed as previously described.4
The output of the second round of RDA was subcloned using a cloning kit (TOPO TA; Invitrogen) to create a minilibrary in a bacterial host. Following the well-established protocol of Weldford et al.,5
2000 clones were randomly picked and then transferred into 96-well plates containing glycerol-based medium for growth and long-term storage at −80°C. The individual inserts from each colony were amplified by using vector-specific primers to generate sufficient material for cDNA microarray printing. Microarrays were prepared in the UCLA Microarray DNA Facility, as previously described.5,6
Probe Labeling and Hybridization to Microarrays
Two micrograms of normal and cd
dog RDA amplicons were labeled with Cy3- and Cy5-dCTP, respectively, using random primers and Klenow enzyme.6
Arrayed cDNAs were first hybridized with these labeled amplicons and 40 of the clones that had the brightest signal with the normal amplicons were sequenced yielding only eight unique fragments. These were mixed and used as a probe for repetitive hybridization of the arrayed library, eliminating in this way a large number of clones and creating a nonredundant set. From this set, only the normal and cd
mRNAs that were differentially expressed were sequenced (ABI Prism 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA).
Microarray Image Acquisition and Data Analysis
The image corresponding to each fluorophore was captured by using an epiconfocal scanner (GMS 418; Affymetrix, Palo Alto, CA) at a resolution of 10 nm and analyzed (Scanalyze software; version 2.44).7
Hybridization signals for each probe were normalized using the Cy5/Cy3 intensity ratio of a housekeeping gene, GAPDH
, which was also spotted onto the glass microarrays.
RT-PCR and Cloning
Total adult mouse retinal RNA was treated to eliminate possible genomic DNA contamination (Turbo DNA-free kit; Ambion, Austin, TX) and then reverse transcribed (Superscript II reverse transcriptase; Invitrogen) with oligo(dT) primers. PCR was performed as follows using a polymerase mix (Advantage 2; BD Biosciences, Franklin Lakes, NJ), 0.2 mM dNTP and 0.2 μM appropriate PCR primers: 94°C for 2 minutes; 35 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 10 minutes. Purified PCR products were cloned into the pCRII vector (TOPO cloning kit; Invitrogen) and sequenced.
Northern Blot Analysis
Two micrograms of polyA+ RNA obtained from human retinal tissue and Y79 retinoblastoma (Y79) cells were electrophoresed on 1.2% denaturing formaldehyde agarose gels and transferred to membranes (Hybond N+; Amersham Biosciences, Piscataway, NJ). Human retinal cDNA fragments amplified from the 5′ (HZB4) or 3′(HZB22) sequences of ZBED4
() were labeled with 32
P-dCTP and used as probes in the hybridizations.6
Quantitative Real-Time RT-PCR
QPCR was performed on first-strand cDNAs as previously described.8
Primers for selected genes were designed using Primer 3 Internet software (http://frodo.wi.mit.edu/provided
in the public domain by the Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA) and synthesized by Invitrogen ().9
Briefly, the QPCR reaction was performed in SYBR Green master mix and the corresponding primer sets, using a quantitative PCR system (MX3000P; Stratagene, La Jolla, CA). The melting curves of PCR products were monitored to ensure that a single melting curve was obtained. For analysis of the real-time PCR data, signals from each sample were normalized to values obtained for a housekeeping gene, β
-actin, which was assayed simultaneously with experimental samples. Primer pairs were subjected to an efficiency test described in ABI’s User Bulletin 2. For those primer pairs with a very close efficiency, duplicate PCR reactions were performed, and the mean of the two reactions was used to calculate the relative gene expression (x
-fold change) between the test and control samples. For this, the comparative threshold cycle (Ct) method (ΔΔ
Ct) was used, which corrects for any difference in the expression of the internal normalization gene (β
-actin). For primer pairs with different efficiencies, the relative expression level of the experimental to the control samples was obtained by using a standard curve for each target gene. This curve was generated with 10-fold serial dilutions of the normal cDNA samples ranging from 0.01 to 100 ng.
Cell Dissociation and Flow Cytometry
Mouse retinas (40 –90 days old) were digested with papain (15 U/mL). Dissociated cells were collected by centrifugation (800 rpm, 10 minutes), resuspended in phosphate-buffered saline (PBS) containing 1% BSA, and incubated with diluted (1:250) FITC-conjugated peanut agglutinin (PNA; Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. The cells were then washed with PBS/1% BSA, adjusted to 1 × 106 cells/mL, immediately sorted by flow cytometry (FACScan; BD Biosciences), and analyzed (Cellquest software; BD Biosciences). Sorted cells were collected by centrifugation and cDNAs were synthesized (One-Step Cells-to-cDNA II kit; Ambion) and subjected to QPCR with primer sets PDEα, ZB-27, PDEα′-1, and mA1 (). All experiments were repeated at least three times, and the results were corroborated using different primer sets: ZB-38, mA2, and PDEα′-2 ().
In Situ Hybridization
In situ hybridization using 8-μ
m frozen sections of human retina embedded in OCT was performed as previously described.10
Two fragments of the human ZBED4
cDNA, one generated with the HZB22 (532 bp) and the other with the HZB4 (557 bp) sets of primers () were subcloned into the pCRII plasmid vector (Invitrogen) for generation of riboprobes. The antisense and sense digoxigenin (DIG)-labeled RNA riboprobes were synthesized with SP6 and T7 RNA polymerases (according to the DIG Labeling Kit protocol; Roche, Indianapolis, IN) and purified by spin columns (NucWay; Ambion). Sections were permeabilized with proteinase K (1 μ
g/mL). Prehybridization was performed at 70°C for 30 minutes in 50% formamide, 5× SSC, 50 μ
g/mL yeast RNA, 50 μ
g/mL heparin, and 1% SDS mixture, and hybridization took place overnight at 70°C in a humidified chamber. Sections were then washed with 5× SSC and 1% SDS in 50% formamide at 70°C, 2× SSC in 50% formamide at 65°C, and Tris-buffered saline-0.1% Tween-20 at room temperature. Sections were incubated overnight at 4°C with alkaline phosphatase-conjugated sheep anti-DIG antibody (1:200; Roche) in 25 mM Tris-HCl (pH 7.5) 140 mM NaCl, 2.7 mM KCl, 1% Tween-20, and 1% sheep serum; washed; and incubated with nitro-blue tetrazolium (NBT) and 5-bromo-4 chloro-3 indolyl phosphate (BCIP) to visualize the mRNA. The reaction was stopped with acidic PBS containing 0.1% Tween-20.
To produce full-length protein, three ZBED4 cDNA fragments were amplified by RT-PCR from mouse retinal RNA using three primer sets containing the desired restriction enzyme recognition sites (KpnI-ZB-SmaI, SmaI-ZB-HindIII, and Pf1M1-ZB-NotI; ). The resultant PCR products were purified with the PCR gel extraction kit (Qiagen) and cloned into the pCRII vector (Invitrogen). All three recombinants were restriction mapped, and those containing the expected insert size were sequenced, digested with the appropriate restriction enzymes, and subcloned in order into the pcDNA4/HisMax A vector between its KpnI/NotI cloning sites. The CMV promoter of this vector drove expression of the ZBED4 fusion protein with the Xpress epitope at its N terminus (see ).
Figure 5 Distribution of expressed ZBED4 in HEK293. (A) ZBED4 expression construct. (B) Immunocytochemical detection of the ZBED4 protein after transient transfection of the expression vector into HEK293 cells. Cells were double stained with antibodies against (more ...)
Transient and Stable Transfections
Y79 retinoblastoma (Y79) and human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown and transfected as previously described,10
using the pcDNA4/HisMax-ZBED4 expression construct and a transfection reagent (PolyFect Transfection Reagent; Qiagen). All experiments included the pcDNA4/HisMax plasmid without insert as an internal normalization control. Cells were harvested 24, 48, and 72 hours after transient transfection for analysis of newly synthesized protein. Linearized plasmid was used to achieve stable transfection of HEK293 cells, and positive clones were selected (Zeocin; Invitrogen).
Generation of Anti-ZBED4 Antibodies
Two ZBED4 peptides containing amino acids 8–25 (N terminus) and 1061–1083 (C terminus) were synthesized and used to immunize rabbits to generate anti-ZBED4 antibodies. These were affinity purified (ProSci, Poway, CA).
Protein Extraction and Immunoblot Analysis
Nuclear and cytoplasmic protein extracts from transfected cells, mouse thymus, and human retinas were prepared with nuclear and cytoplasmic extraction reagents (NE-PER; Pierce Biotechnology, Rockford, IL). Fifty micrograms of extracted proteins were separated by SDS-PAGE on 7.5% gels (Pierce Biotechnology). Blots were incubated with primary antibodies (1:1000 dilution) and secondary anti-rabbit IgG antibodies labeled with alkaline phosphatase (1:5000 dilution; Vector Laboratories). Western blots were visualized with either of two kits (the Amplified-Alkaline Phosphatase kit; BioRad Laboratories, Hercules, CA, or the Enhanced Chemiluminescence kit [ECL]; Amersham).
Three siRNA sequences were chosen from the ZBED4 open reading frame (GenBank Accension No. NM014838 and gene ID9889) to target ZBED4: nt 2589–2601, 5′-GGUAUGUAUGAUAAUGUGA-3′ (S1); nt 548–566, 5′-GGAAGAUGAUGAUGGAAUU-3′ (S2); and nt 608–626, 5′-GGAGGACAUGAAGCAGACA-3′ (S3). The dsRNA nucleotides were chemically synthesized and annealed by Ambion, Inc. GAPDH siRNA was used as the positive control; the three negative controls were (1) cells transfected without addition of siRNA, to determine any nonspecific effects that could be caused by the transfection reagent; (2) cells transfected with a nonsilencing siRNA with no homology to any known mammalian gene, to determine whether changes in gene expression were nonspecific; and (3) nontransfected cells to allow measurement of the basal level of gene expression.
The day before transfection, 2× 105 HEK293 or Y79 cells were seeded into six-well plates with culture medium containing serum and antibiotics. The next day, 12 μL of transfection reagent (HiPerfect; Invitrogen) was added to 300 ng siRNAs in 100 μL culture medium without serum. The samples were incubated for 5 to 10 minutes at room temperature to allow the formation of transfection complexes. The complexes were added drop-wise onto 50% to 80% confluent cells to a final siRNA concentration of 10 nM. Transfected cells were harvested after 24, 48, and 72 hours to monitor gene silencing by QPCR and Western blot analysis.
Immunostaining of Cultured Cells
Transfected HEK293 or Y79 cells on coverslips were permeabilized with 100% methanol for 6 minutes at −20°C, rinsed three times in PBS, blocked with 3% BSA in PBS containing 0.1% Triton X-100 (PBST) for 45 minutes, and incubated for 2 hours with N terminus anti-ZBED4 rabbit polyclonal antibody (1:200 dilution) and subsequently 1 hour with fluorescein- or rhodamine-conjugated goat anti-rabbit antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Next, the transfected cells were incubated 1 hour with a mouse monoclonal antibody (1:100 dilution, anti-Xpress-FITC; Invitrogen). The cells were washed three times in PBST, stained with propidium iodide (PI) or with 4′, 6-diamidino-2-phenylindole (DAPI) for nuclei detection and viewed by fluorescence microscopy.
Immunostaining of Human Retinal Sections
Human retinas were embedded in OCT (Sakura Finetek USA., Inc., Torrance, CA). Frozen sections (8 μm) were cut on a cryostat (Leica CM1850; McBain Instruments, Chatsworth, CA). Before immunofluorescent staining, the sections were fixed for 5 minutes at room temperature in 4% paraformaldehyde in PBS, washed with PBS and reacted with the same primary and secondary antibodies described in the prior section. To show the colocalization of ZBED4 with cone cells and Müller cells, sections were also incubated with rhodamine-conjugated PNA (1:250, 1 hour; Vector Laboratories, catalog no. RL-1072) and goat anti-human vimentin (Sigma-Aldrich, St. Louis, MO, catalog no. V-4630), respectively, along with the corresponding secondary antibodies. Images were obtained with a digital camera (MagnaFire; Optronics, Goleta, CA) attached to a microscope (model BX40; Olympus USA, Melville, NY) and were merged using the camera’s software (MagnaFire 2.1; Optronics). Negative controls (preimmune serum instead of primary antibody or omission of primary antibody) were included with each experiment. ZBED4 rabbit polyclonal antibody pre-absorbed with ZBED4 peptide was used to stain some tissues.