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Evidence for Baseline Retinal Pigment Epithelium Pathology in the Trp1-Cre Mouse

  • Aristomenis Thanos
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Yuki Morizane
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Yusuke Murakami
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Andrea Giani
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Dimosthenis Mantopoulos
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Maki Kayama
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Mi In Roh
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Norman Michaud
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Basil Pawlyk
    Affiliations
    Berman-Gund Laboratory For the Study of Retinal Degenerations, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Michael Sandberg
    Affiliations
    Berman-Gund Laboratory For the Study of Retinal Degenerations, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Lucy H. Young
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Joan W. Miller
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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  • Demetrios G. Vavvas
    Correspondence
    Address reprint requests to Demetrios G. Vavvas, M.D., Ph.D., Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles St., Boston, MA, 02114
    Affiliations
    Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
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Open AccessPublished:March 19, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.01.017
      The increasing popularity of the Cre/loxP recombination system has led to the generation of numerous transgenic mouse lines in which Cre recombinase is expressed under the control of organ- or cell-specific promoters. Alterations in retinal pigment epithelium (RPE), a multifunctional cell monolayer that separates the retinal photoreceptors from the choroid, are prevalent in the pathogenesis of a number of ocular disorders, including age-related macular degeneration. To date, six transgenic mouse lines have been developed that target Cre to the RPE under the control of various gene promoters. However, multiple lines of evidence indicate that high levels of Cre expression can be toxic to mammalian cells. In this study, we report that in the Trp1-Cre mouse, a commonly used transgenic Cre strain for RPE gene function studies, Cre recombinase expression alone leads to RPE dysfunction and concomitant disorganization of RPE layer morphology, large areas of RPE atrophy, retinal photoreceptor dysfunction, and microglial cell activation in the affected areas. The phenotype described herein is similar to previously published reports of conditional gene knockouts that used the Trp1-Cre mouse, suggesting that Cre toxicity alone could account for some of the reported phenotypes and highlighting the importance of the inclusion of Cre-expressing mice as controls in conditional gene targeting studies.
      The retinal pigment epithelium (RPE) is a multifunctional, cuboidal monolayer of cells that separates the retinal photoreceptors from the choroid. The RPE is an essential component of the vertebrate retina and performs diverse functions, including the formation of the outer blood retinal barrier, the phagocytosis of photoreceptor outer disk segments, and the regeneration of 11-cis retinal for the visual cycle.
      • Zinn K.M.
      • Benjamin-Henkind J.V.
      Anatomy of the human retinal pigment epithelium The Retinal Pigment Epithelium.
      • Strauss O.
      The retinal pigment epithelium in visual function.
      Dysfunction of the RPE has been linked with a variety of ocular disorders, including age-related macular degeneration and retinitis pigmentosa, among others.
      The Cre-loxP recombination system has been instrumental in dissecting the spatial and temporal functions of many important genes involved in development, physiology, or disease through the generation of organ- or cell-specific knockout animals. It is an elegant technique to bypass lethal or severe developmental defects that result from systemic ablation of essential genes.
      • Nagy A.
      Cre recombinase: the universal reagent for genome tailoring.
      Cre recombinase is a 38-kDa protein that catalyzes the recombination between two of its loxP recognition sites, a 34-bp sequence consisting of a core 8-bp sequence and two 13-bp palindromic flanking sequences.
      • Hamilton D.L.
      • Abremski K.
      Site-specific recombination by the bacteriophage P1 lox-Cre system Cre-mediated synapsis of two lox sites.
      However, several lines of evidence indicate that Cre expression can be toxic in mammalian cells because it can cleave mammalian DNA in a nonspecific manner at sequences that share limited homology with the 34-bp loxP sequence.
      • Thyagarajan B.
      • Guimaraes M.J.
      • Groth A.C.
      • Calos M.P.
      Mammalian genomes contain active recombinase recognition sites.
      Pseudo/cryptic-loxP sites that occur naturally in Escherichia coli and in yeast and mammalian genomes serve as low-affinity substrates for Cre recombinase,
      • Sauer B.
      Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance.
      • Sauer B.
      Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome.
      • Sternberg N.
      • Hamilton D.
      • Hoess R.
      Bacteriophage P1 site-specific recombination, II: recombination between loxP and the bacterial chromosome.
      and continuous exposure to high concentrations of the enzyme triggers cleavage and recombination between these sites. These “illegitimate” recombinations can result in growth inhibition, cell cycle arrest, DNA strand breaks, or even gross chromosomal aberrations.
      • Loonstra A.
      • Vooijs M.
      • Beverloo H.B.
      • Allak B.A.
      • van Drunen E.
      • Kanaar R.
      • Berns A.
      • Jonkers J.
      Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells.
      • Schmidt E.E.
      • Taylor D.S.
      • Prigge J.R.
      • Barnett S.
      • Capecchi M.R.
      Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids.
      • de Alboran I.M.
      • O'Hagan R.C.
      • Gartner F.
      • Malynn B.
      • Davidson L.
      • Rickert R.
      • Rajewsky K.
      • DePinho R.A.
      • Alt F.W.
      Analysis of C-MYC function in normal cells via conditional gene-targeted mutation.
      Here, we report the phenotype induced by Cre expression in the RPE of the Trp1-Cre transgenic mouse strain, where Cre is expressed under the control of tyrosinase-related protein 1 (Trp1/Tyrp1, hereafter referred as Trp1) gene promoter. Trp1 belongs to the tyrosinase family of proteins, which are uniquely expressed in melanin-synthesizing cells, including the RPE. Trp1 is a glycoprotein located in the melanosomal membrane that acts within the context of a series of reactions in the melanogenic pathway to control melanin production in melanosomes.
      • Kobayashi T.
      • Hearing V.J.
      Direct interaction of tyrosinase with Tyrp1 to form heterodimeric complexes in vivo.
      In this study we describe damage to RPE and retina in the Trp1-Cre mouse that includes drastic changes in gross cellular and ultrastructural morphology, irregularities in gene expression, and electroretinogram (ERG) abnormalities; these alterations are not exhibited in their littermate controls.

      Materials and Methods

      Animals

      All animal experiments followed the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of Massachusetts Eye and Ear Infirmary. The transgenic mouse line expressing Cre recombinase under the control of the Trp1 promoter was used for the experiments.
      • Mori M.
      • Metzger D.
      • Garnier J.M.
      • Chambon P.
      • Mark M.
      Site-specific somatic mutagenesis in the retinal pigment epithelium.
      Genotyping of the Trp1-Cre transgenic allele was performed with the following primer set: Trp1-CreF, 5′-GCGGTCTGGCAGTAAAAACTATC-3′, and Trp1-CreR, 5′-GTGAAACAGCATT GCTGTCACTT-3′, along with an internal control primer set, 5′-CTAGGCCACAG AATTGAAAGATCT-3′ and 5′-GTAGGTGGAAATTCTAGCATCATCC-3′, which produced amplicons of 102 and 324 bp, respectively (see Supplemental Figure S1A at http://ajp.amjpathol.org). The Trp1-Cre mouse strain was backcrossed for at least 10 generations with C57Bl/6 mice. All mice harboring the Trp1-Cre transgene were genotyped for the retinal degeneration 1 mutation,
      • Gimenez E.
      • Montoliu L.
      A simple polymerase chain reaction assay for genotyping the retinal degeneration mutation (Pdeb(rd1)) in FVB/N-derived transgenic mice.
      and only mice that did not carry the mutation were used for the experiments. A minimum of six to eight animals were used for each experiment. Genomic DNA from FVB/N mice was obtained from The Jackson Laboratories (Bar Harbor, ME) and used as a positive control for the retinal degeneration 1 mutation (see Supplemental Figure S1B at http://ajp.amjpathol.org). Littermate mice not carrying the Cre transgene were used as controls for all experiments. The ROSA26R reporter gene mouse was obtained from The Jackson Laboratories.
      • Soriano P.
      Generalized lacZ expression with the ROSA26 Cre reporter strain.
      Anesthesia was achieved by intraperitoneal injection of 50 mg/kg ketamine hydrochloride (Phoenix Pharmaceutical, Inc., St. Joseph, MO) and 10 mg/kg xylazine (Phoenix Pharmaceutical, Inc.), and pupils were dilated with topical 0.5% tropicamide (Alcon, Humacao, Puerto Rico).

      Immunohistochemistry of RPE Flat Mounts

      Under deep anesthesia mice were perfused through the left ventricle with 10 mL of PBS followed by 10 mL of 4% paraformaldehyde. Eyes were enucleated, and the anterior segment and retina were removed under a dissecting microscope. Special attention was paid during the removal of the retina to avoid damage of the underlying RPE. Four relaxing radial incisions were made, and the remaining RPE-choroid-sclera complex was flat mounted on a glass slide. Flat mounts were air-dried for 10 minutes and incubated for 1 hour with blocking solution (5% donkey serum, 0.3% bovine serum albumin, 0.3% Triton-X). Primary antibodies were incubated overnight at 4°C in a moisture chamber. A full list of antibodies as well as their working concentrations is shown in Table 1.
      Table 1Antibodies Used in This Study
      AntigenHostImmunoblotImmunostainingSupplier
      RPE 65Rabbit1:1000Abcam
      β-cateninMouse1:20001:100Millipore
      Cre-recombinaseRabbit1:400Millipore
      FITC-ZO1Goat1:100Invitrogen
      Iba-1Rabbit1:100Wako
      β-tubulinRabbit1:2000Cell Signaling

      X-Gal Staining for β-Galactosidase Activity

      For whole-mount X-Gal staining, eyes were enucleated and fixed for 7 minutes in 4% paraformaldehyde. After fixation, eyes were washed thoroughly three times with wash buffer (0.1 mol/L sodium phosphate, 2 mmol/L MgCl2, 0.01% deoxycholate, 0.02% Nonidet P-40). Immediately after washing, eyes were placed in prewarmed (37°C) X-Gal staining solution (5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6-6H2O diluted in wash buffer) and X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 1 mg/mL. Eyes were protected from light and incubated at 37°C overnight. For tissue sections, eyes were enucleated and embedded into optimal temperature cutting medium (Tissue-Tek OCT; Sakura, Alphen aan den Rijn, The Netherlands). Serial sections of 10 μm were cut using a cryostat (Leica Microsystems, Buffalo Grove, IL) and assayed for β-galacatosidase activity, as described above. To better visualize X-gal staining in the RPE, pigment was bleached (after X-gal staining) by incubation in KMnO4 (0.25% in water) for 35 minutes at room temperature (protected from light) and subsequent incubation in oxalic acid (1% in water) for 20 minutes at room temperature.

      Fluorescein Angiography

      Fluorescein angiography (FA) was performed with a commercial camera and imaging system (TRC 50 VT camera and IMAGEnet 1.53 system; Topcon, Paramus, NJ). Photographs were captured with a 20-diopter lens in contact with the fundus camera lens after intraperitoneal injection of 0.1 mL of 2% fluorescein sodium (Akorn, Decatur, IL).

      ERG Analysis

      ERGs were recorded as previously described.
      • Sun X.
      • Pawlyk B.
      • Xu X.
      • Liu X.
      • Bulgakov O.
      • Adamian M.
      • Sandberg M.A.
      • Khani S.C.
      • Tan M.H.
      • Smith A.J.
      • Ali R.R.
      • Li T.
      Gene therapy with a promoter targeting both rods and cones rescues retinal degeneration caused by AIPL1 mutations.
      Briefly, after an overnight dark adaptation mice were anesthetized with sodium pentobarbital at 80 mg/kg given intraperitoneally. Their pupils were dilated with 0.2% phenylephrine and 0.02% cyclopentolate hydrochloride. Full-field, rod-dominant (>95%) ERGs were elicited in the dark with 10-μsecond flashes of white light (1.37×105 cd/m2) presented at 1-minute intervals in a Ganzfeld dome. Light-adapted, cone responses were elicited in the presence of a 41-cd/m2 rod-desensitizing white background with the same flashes (1.37×105 cd/m2 presented at 1 Hz). Cone responses were collected with signal averaging.

      Electron Microscopy

      Eyes were enucleated under deep anesthesia, and the globe was cleaned of all extraneous tissue then rinsed with saline. The globe was immediately placed into fixative consisting of 2.5% glutaraldehyde and 2% formaldehyde in 0.1 mol/L cacodylate buffer with 0.08 mol/L CaCl2 at 4°C. After a short 10- to 15-minute fixation, the eye was bisected at the limbus, the anterior segment was separated from the posterior segment, and the parts to be examined were placed back in the fixative. Within 24 hours of enucleation, the tissue was washed in 0.1 mol/L cacodylate buffer and stored at 4°C. The tissue was postfixed for 1.5 hours in 2% aqueous OsO4. Tissue was dehydrated in graded ethanols, transitioned in propylene oxide, infiltrated with propylene oxide and epon mixtures (TAAB 812 resin; Marivac, Quebec, QC, Canada), embedded in epon, and cured for 48 hours at 60°C. One-micron sections were cut on a Leica Ultracut UCT and stained with 1% toluidine blue in 1% borate buffer. For transmission electron microscopy observation, thin sections were cut at 70 to 90 nm and stained with saturated, aqueous uranyl acetate and Sato's lead stain. Examination was done on a Philips CM-10 electron microscope (Philips Healthcare, Amsterdam, The Netherlands).

      Animal Spectral Domain Optical Coherence Tomography

      Optical coherence tomography was performed with a spectral domain optical coherence tomography system (Bioptigen Inc., Durham, NC) as previously described.
      • Giani A.
      • Thanos A.
      • Roh M.I.
      • Connolly E.
      • Trichonas G.
      • Kim I.
      • Gragoudas E.
      • Vavvas D.
      • Miller J.W.
      In vivo evaluation of laser-induced choroidal neovascularization using spectral-domain optical coherence tomography.
      Briefly, a volume analysis centered on the optic nerve head was performed, using 100 horizontal, raster, and consecutive B-scan lines, each one composed by 1200 A-scans. The volume size was 1.6 × 1.6 mm. Total retinal thickness and outer nuclear layer thickness was assessed at distances of 500 μm, 400 μm, and 300 μm from the optic nerve head (nasally and temporally) and at 200 μm above and 400 μm below the optic nerve head.

      Western Blot Analysis

      The choroid-RPE tissue from Trp1-Cre mice and respective controls was separated from the retina and homogenized in lysis buffer,
      • Vavvas D.
      • Apazidis A.
      • Saha A.K.
      • Gamble J.
      • Patel A.
      • Kemp B.E.
      • Witters L.A.
      • Ruderman N.B.
      Contraction-induced changes in acetyl-CoA carboxylase and 5′-AMP-activated kinase in skeletal muscle.
      supplemented with a mixture of proteinase inhibitors (Complete Mini; Roche Diagnostics, Basel, Switzerland). The samples were centrifuged (14,000 rpm for 30 minutes at 4°C), and supernatant fluids were collected. Protein concentration was assessed with the bicinchoninic acid protein assay (Pierce, Rockford, IL). Thirty micrograms of protein per sample were separated in a 4% to 20% gradient SDS-PAGE (Invitrogen Corporation, Carlsbad, CA), and the proteins were electroblotted onto polyvinylidene difluoride membranes. After 20 minutes of incubation in blocking solution (Starting Block T20; Thermo Scientific, Waltham, MA), membranes were incubated with primary antibodies overnight at 4°C. Table 1 includes a full list of antibodies and their working concentrations. Peroxidase-labeled secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) were used, and proteins were visualized with enhanced chemiluminescence technique (Amersham Pharmacia Biotech).

      ELISA

      Analysis of IL-10 production was performed using a quantitative ELISA kit (R&D Systems, Minneapolis, MN). After perfusion with 10 mL of PBS, choroid-RPE lysates from wild-type and Trp1-Cre mice were collected and assayed for IL-10 protein levels according to the manufacturer's instructions. Values were normalized to lysate protein levels.

      Statistical Analysis

      Values are expressed as mean ± SEM (unless specified), and statistical analysis was performed with an unpaired Student's t-test or with the non-parametric Mann-Whitney U test for unequal variances.

      Results

      Expression Pattern of Cre-Recombinase in the Trp1-Cre Mouse

      To evaluate the expression pattern of Cre recombinase in the Trp1-Cre transgenic mouse strain, adult 2-month-old Trp1-Cre mice were crossed with the ROSA26 reporter gene mouse to yield a Trp1-Cre;ROSA26R line. Upon Cre gene expression, the recombinase activity results in the excision of a loxP-flanked STOP sequence that prevents expression of a lacZ gene.
      • Soriano P.
      Generalized lacZ expression with the ROSA26 Cre reporter strain.
      Thus, β-galactosidase staining reflects Cre recombinase activity in vivo. We confirmed that the Trp1-Cre mouse provided robust Cre expression in the RPE (Figure 1C). To estimate the percentage of Cre-expressing cells, X-Gal staining and subsequent melanin bleaching were performed on whole eyecups of Trp1-Cre;ROSA26R mice. A variable degree of mosaicism in Cre expression was observed, ranging from 60% to 90% of total RPE cells (Figure 1F; see also Supplemental Figure S1C at http://ajp.amjpathol.org). Ectopic Cre expression in other ocular tissues was evident such as the ciliary margin of the retina (Figure 1, A, B, and E), ciliary pigment epithelium (Figure 1B), and optic nerve stalk (see Supplemental Figure S1D at http://ajp.amjpathol.org). ROSA26R eyecups incubated with X-Gal staining solution did not show any β-galactosidase activity. (Figure 1, D, G, and H) Taken together, these data indicate that the expression pattern of our Trp1-Cre strain is consistent with the one described by Mori et al
      • Mori M.
      • Metzger D.
      • Garnier J.M.
      • Chambon P.
      • Mark M.
      Site-specific somatic mutagenesis in the retinal pigment epithelium.
      in the initial description of the Trp1-Cre mouse.
      Figure thumbnail gr1
      Figure 1β-galactosidase activity in the Trp1-Cre;ROSA26R line. Sagittal frozen sections showing Cre expression in the ciliary margin of the retina (A), with magnified images of the ciliary body (B) and RPE (C); X-Gal staining is blue (arrows). D: ROSA26R control RPE. Representative RPE flat mounts from Trp1-Cre;ROSA26R (E and F) mice showing the extent of Cre expression in the RPE and peripheral retina, which was folded over for better visualization of X-Gal staining (blue; arrows). Melanin bleaching (F–H) to identify the percentage of Cre-expressing cells. No β-galactosidase activity was observed in ROSA26R controls incubated with X-Gal staining solution (D, G, and H). Scale bars: 100 μm (A), 150 μm (B), 50 μm (C and D), 500 μm (E–H).

      Severely Altered RPE Morphology in the Trp1-Cre Mouse

      Intercellular tight junctions are critical for the maintenance of RPE monolayer integrity. Adherens proteins, such as β-catenin, are important for intercellular adhesion and preservation of the epithelial morphology, whereas tight junction proteins, such as zona occludens (ZO)-1, have a fundamental role for the formation and function of outer blood retinal barrier.
      • Rizzolo L.J.
      Polarity and the development of the outer blood-retinal barrier.
      To examine the morphology of the RPE layer in the Trp1-Cre mouse, immunofluorescent staining was performed on flat mounts with the use of a monoclonal antibody against the adherens junction protein β-catenin. In Trp1-Cre mice, the majority of the RPE monolayer had lost its classic honeycomb appearance, and the cells were enlarged, polygonal, and dysmorphic compared with wild-type controls (Figure 2, A–F). Quantitative analysis showed a dramatic decrease in RPE cell density (number of cells per unit area; Figure 2G). Average cell size and perimeter were found to be significantly higher in Trp1-Cre mice compared with wild types (Figure 2, H and I). Similar results were obtained after immunostaining with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against ZO-1 (see Supplemental Figure S2A at http://ajp.amjpathol.org). Furthermore, although β-catenin is mostly associated with the cell membrane, an aberrant cytoplasmic distribution was evident, indicating a disturbance in its characteristic membranous association (Figure 2, D and F). The latter was accompanied by a reduction of protein expression as shown by Western blot analysis on 2-month-old choroid-RPE lysates (Figure 2, J and K). Finally, immunofluorescent staining with a mouse monoclonal antibody against Cre recombinase found nuclear localization of the protein as expected (Figure 2F).
      Figure thumbnail gr2
      Figure 2Morphologic abnormalities of RPE monolayer of the Trp1-Cre mouse. Immunohistochemical staining for the junctional protein β-catenin showed severe disorganization of RPE cells (B and D) with loss of its classic honeycomb appearance (A and C). Substantial loss of β-catenin membranous association (D and F arrows) was also observed compared with wild-type controls (C and E). Significant decrease in cell density (G), increase in cell size (H), and cell perimeter (I) in Trp1-Cre mice. J: Western blot analysis of choroid-RPE protein lysates from wild-type and Trp1-Cre mice showed significant decrease in β-catenin protein expression levels. K: Densitometric analysis of β-catenin expression. Error bars indicate SD (G, H, and I) and SEM (K). Scale bars: 50 μm (A and C), 200 μm (B, D–F). **P < 0.01, ***P < 0.001.

      Pigmentary Defects in the RPE of the Trp1-Cre Mouse

      Macroscopic examination of RPE flat mounts from Trp1-Cre mice found wedge-shaped areas of pigmentary defects that were extending from the mid-periphery until the ora serrata compared with control (Figure 3, A and B). To further examine this finding, FA was performed in adult 1-month-old animals. The pigmentary defects observed previously corresponded to areas of hyperfluoresence that increased in intensity over time but not in size (window defect) (Figure 3, C–H). These defects were bilateral and interestingly found at all times in similar position in the periphery of the eye (2 and 10 o'clock meridians). In addition, late-phase angiograms of Trp1-Cre mice found diffuse background hyperfluorescence (Figure 3, G and H). Next, Trp1-Cre and wild-type littermate controls were perfused with rhodamine-conjugated concanavalin-A lectin and counterstained the overlying RPE layer with a FITC-conjugated antibody against ZO-1 (Figure 3, I–N). As expected, in wild-type animals the RPE layer precluded visualization of the underlying choroid because of its dense melanin content (Figure 3, I–K), whereas in Trp1-Cre mice the choroidal vasculature was visible beneath the RPE, indicating a defect in its pigmentation (Figure 3, L–N), which may account, at least in part, for the diffuse background hyperfluorescence observed on FA. This was further supported from 1-μm semithin sections stained with toluidine blue, where the RPE layer was flattened with large discontinuities in its pigmentation (Figure 3, O–Q).
      Figure thumbnail gr3
      Figure 3Pigmentary defects in Trp1-Cre mice. A and B: Macroscopic examination of RPE flat mounts from wild-type and Trp1-Cre mice showed large wedge-shaped areas of depigmentation (arrows). C–H: Early- and late-phase FA showed large areas of hyperfluorescence at 10 o'clock (G) and 2 o'clock (H) meridians. I–N: Perfusion with rhodamine-conjugated Concanavalin A lectin and counterstaining with FITC-ZO-1. In wild-type mice, the melanin content of the RPE blocked the fluorescent signal from the choroid. In Trp1-Cre mice visualization of the underlying large bore capillary bed of the choroid can be seen because of abnormalities in its pigmentation. O–Q: Large areas of depigmented and flattened RPE cells (enlarged in P; arrows) were seen on 1-μm toluidine blue sections, compared with the uniform shape of wild-type RPE cells (Q). Scale bars: 500 μm (A and B), 50 μm (I–N), 100 μm (P and Q).

      Morphologic Abnormalities in the RPE of the Trp1-Cre Mice

      Next, the morphology of the RPE layer was assessed by transmission electron microscopic analysis of adult 2-month-old wild-type and Trp1-Cre mice (Figure 4). A substantial decrease was observed in the thickness of RPE cells of Trp1-Cre mice with either loss or disorganization of the apical villi, which did not integrate with the outer segments of photoreceptors (Figure 4, D–I). In addition, a reduction of basal laminar infoldings was noted (Figure 4, E–H). Melanin granules were larger (Figure 4, E and F) and irregularly shaped (Figure 4, E and H) compared with the elliptical or spherical shape of wild-type controls (Figure 4, A–C). RPE cells with little or no melanin granules were also evident (Figure 4, D, G, and I). In line with the initial description of the Trp1-Cre mouse, there were no abnormalities in the shape of choroidal melanocytes (Figure 4, B, E–G), which do not express Cre recombinase.
      • Mori M.
      • Metzger D.
      • Garnier J.M.
      • Chambon P.
      • Mark M.
      Site-specific somatic mutagenesis in the retinal pigment epithelium.
      Figure thumbnail gr4
      Figure 4Ultrastructural analysis of the RPE of littermate controls (A–C) and the Trp1-Cre mouse (D–I). Abnormalities in the number (D and I), shape (E), and size (F and H) of RPE melanin granules were seen in Trp1-Cre mice compared with the fusiform shape of wild-type controls (A–C). Notice the unusually large melanin granule (asterisk; F). The apical villi (AV) were collapsed and did not integrate with photoreceptors, as in wild-type RPE. Microglial cell (M) with phagocytic granules in the subretinal space (E, outline). AV, apical villi, BLI, basal laminar infolding; BRM, Bruch's membrane; CC, choriocapillaris; CH, choroid; M, macrophage/microglial cell; POS, photoreceptor outer segment. n = 6 eyes per group. Scale bars: 1 μm (A, B, D, and I), 2 μm (C), 2.5 μm (E, F, and G).

      Retinal Abnormalities in Trp1-Cre Mice

      Given the critical role of the RPE in the maintenance and function of the photoreceptor cell layer, retinal function of Trp1-Cre mice was examined. Full-field dark-adapted (rod) and light-adapted (cone) ERGs were recorded from 4-month-old animals (n = 4 for each group). Both rod and cone responses were significantly reduced in Trp1-Cre mice compared with the ERG responses obtained from wild-type mice counterparts (Figure 5A). Consistent with the abnormal ERG data, a pronounced decrease in total retinal thickness (Figure 5, B and C) and outer nuclear layer thickness (Figure 5, B and D) was seen in Trp1-Cre mice as examined in vivo with spectral domain optical coherence tomography. Finally, Western blot analysis of choroid-RPE lysates for RPE65, a protein abundantly expressed in the RPE that plays a critical role in retinoid processing, showed a significant reduction in its expression level compared with wild-type animals, suggesting possible deficits in the visual cycle (Figure 5E).
      Figure thumbnail gr5
      Figure 5Retinal abnormalities in Trp1-Cre mice. A: Representative dark adapted and light adapted responses in 4-month-old control and Trp1-Cre mice. Amplitudes of both a-waves and b-waves were reduced in Trp1-Cre mice. n = 4. P < 0.001 for both a and b waves. B: In vivo evaluation with spectral domain optical coherence tomography at the level of optic nerve (arrow) and 200 μm above (arrowhead) and measurement of total retinal thickness (blue arrow) and outer nuclear layer thickness (orange arrow). C and D: Significant decrease in total retinal thickness and outer nuclear layer thickness was seen in Trp1-Cre mice. E: Western blot analysis of choroid-RPE lysates for RPE65 protein expression levels (n = 6 eyes per group).

      Microglial Cell Activation in the Trp1-Cre Mouse

      The retinal microglia constitute the resident macrophages of the retina and are among the first cells to respond after tissue injury.
      • Langmann T.
      Microglia activation in retinal degeneration.
      Under normal conditions, the retinal microglia are distributed throughout the retinal layers with the notable exception of the subretinal space, which is considered an immune-privileged site.
      • Jiang L.Q.
      • Jorquera M.
      • Streilein J.W.
      Subretinal space and vitreous cavity as immunologically privileged sites for retinal allografts.
      To investigate whether Cre-mediated RPE damage triggers microglial cell activation, immunofluorescent staining was performed on RPE flat mounts with the use of an antibody against the macrophage/microglial cell marker Iba-1, a calcium-binding protein involved in membrane ruffling and phagocytosis, which is significantly up-regulated during microglial cell activation.
      • Kanazawa H.
      • Ohsawa K.
      • Sasaki Y.
      • Kohsaka S.
      • Imai Y.
      Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma-dependent pathway.
      No microglial cells were detected in the subretinal space of wild-type 2-month-old mice (see Supplemental Figure S3, A–C, at http://ajp.amjpathol.org). By contrast, immunofluorescent staining of RPE flat mounts from Trp1-Cre mice showed a dramatic accumulation of microglial cell in the subretinal space, mostly at the areas of the damaged RPE (Figure 6, A–F; see also Supplemental Figure S3, D–F, at http://ajp.amjpathol.org). Furthermore, microglial cells acquired an amoeboid-like morphology with short processes (Figure 6H) indicative of their phagocytic-activated state. Moreover, their cell bodies co-localized with cellular debris stained positive for FITC-ZO-1 that was most likely originating from phagocytosed RPE cells (Figure 6, G–I; see also Supplemental Figure S3, D–F, at http://ajp.amjpathol.org). Finally, microglial cells were intimately associated with areas of dysfunctional RPE cell membranes or areas of RPE remodeling, as identified from the absence of ZO-1 immunostaining of RPE cell membranes or the structural changes of surrounding RPE cells, respectively (Figure 6, J–L; see also Supplemental Figure S, 3K–M, at http://ajp.amjpathol.org).
      Figure thumbnail gr6
      Figure 6Microglial cell activation and migration to the subretinal space in the Trp1-Cre mouse. A–F: Iba-1-positive microglial cells were mostly confined to the areas of injured RPE cells. G–I: Cellular debris stained positive for FITC-ZO1 co-localized with cell bodies of activated, amoeboid-shaped microglial cells (arrows). J–L: Microglial cells were commonly associated with areas of dysfunctional RPE cell membranes, as indicated from the absence of ZO-1 immunostaining. Scale bars: 200 μm (A–C), 100 μm (D–F), 25 μm (G–L).

      Discussion

      The present study describes a phenotype in the Trp1-Cre mice characterized by dramatic changes in the morphology of the RPE monolayer, irregularities in its pigmentation, retinal dystrophy, and concomitant activation of the retinal microglia in the affected areas. The described phenotype had 100% penetrance with patchy distribution and was observed in both male and female homozygous mice for the Trp1-Cre transgene. In the Trp1-Cre strain, Cre expression in the RPE starts from embryonic day 10.5 (time of RPE differentiation) to postnatal day 12,
      • Mori M.
      • Metzger D.
      • Garnier J.M.
      • Chambon P.
      • Mark M.
      Site-specific somatic mutagenesis in the retinal pigment epithelium.
      and significant changes in RPE monolayer appearance were evident also on postnatal day 14, indicating that Cre may have exerted its toxic effect even before this time point (see Supplemental Figure S2B at http://ajp.amjpathol.org).
      It is known that the insertion of a Cre transgene or any other transgene may affect the function of the upstream gene promoter chosen to drive transgene expression. For instance, CD19-Cre mice, which are used to evaluate gene function in B cells, show 50% decrease in the levels of CD19 expression compared with wild-type B cells.
      • Schmidt-Supprian M.
      • Rajewsky K.
      Vagaries of conditional gene targeting.
      Similarly, rat insulin II promoter-Cre mice, expressing Cre in pancreatic β cells, are glucose intolerant.
      • Lee J.Y.
      • Ristow M.
      • Lin X.
      • White M.F.
      • Magnuson M.A.
      • Hennighausen L.
      RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function.
      Therefore, the abnormalities in RPE pigmentation and melanosome shape we observed in the Trp1-Cre mouse and previously reported by two other studies
      • Marneros A.G.
      • Fan J.
      • Yokoyama Y.
      • Gerber H.P.
      • Ferrara N.
      • Crouch R.K.
      • Olsen B.R.
      Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function.
      • Mori M.
      • Metzger D.
      • Picaud S.
      • Hindelang C.
      • Simonutti M.
      • Sahel J.
      • Chambon P.
      • Mark M.
      Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium.
      may be attributed to abnormalities in the function of Trp1 gene promoter, which drives Cre expression in this particular strain and is heavily involved in melanin synthesis under normal conditions.
      • Kobayashi T.
      • Urabe K.
      • Winder A.
      • Jimenez-Cervantes C.
      • Imokawa G.
      • Brewington T.
      • Solano F.
      • Garcia-Borron J.C.
      • Hearing V.J.
      Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis.
      Findings described in this study suggest that some of the phenotypes described in prior RPE-specific knockout studies that used Trp1-Cre mice may have been a combination of the targeted loxP gene excision and/or the Cre transgene presence toxicity itself (Table 2) .
      • Marneros A.G.
      • Fan J.
      • Yokoyama Y.
      • Gerber H.P.
      • Ferrara N.
      • Crouch R.K.
      • Olsen B.R.
      Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function.
      • Mori M.
      • Metzger D.
      • Picaud S.
      • Hindelang C.
      • Simonutti M.
      • Sahel J.
      • Chambon P.
      • Mark M.
      Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium.
      • Westenskow P.
      • Piccolo S.
      • Fuhrmann S.
      Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression.
      • Ruiz A.
      • Ghyselinck N.B.
      • Mata N.
      • Nusinowitz S.
      • Lloyd M.
      • Dennefeld C.
      • Chambon P.
      • Bok D.
      Somatic ablation of the Lrat gene in the mouse retinal pigment epithelium drastically reduces its retinoid storage.
      • Kim J.W.
      • Kang K.H.
      • Burrola P.
      • Mak T.W.
      • Lemke G.
      Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium.
      • Fujimura N.
      • Taketo M.M.
      • Mori M.
      • Korinek V.
      • Kozmik Z.
      Spatial and temporal regulation of Wnt/beta-catenin signaling is essential for development of the retinal pigment epithelium.
      • Schouwey K.
      • Aydin I.T.
      • Radtke F.
      • Beermann F.
      RBP-Jkappa-dependent Notch signaling enhances retinal pigment epithelial cell proliferation in transgenic mice.
      For instance, retinal dystrophy with abnormal ERG responses and decreased RPE65 expression have been reported by two independent studies
      • Mori M.
      • Metzger D.
      • Picaud S.
      • Hindelang C.
      • Simonutti M.
      • Sahel J.
      • Chambon P.
      • Mark M.
      Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium.
      • Ruiz A.
      • Ghyselinck N.B.
      • Mata N.
      • Nusinowitz S.
      • Lloyd M.
      • Dennefeld C.
      • Chambon P.
      • Bok D.
      Somatic ablation of the Lrat gene in the mouse retinal pigment epithelium drastically reduces its retinoid storage.
      that used the Trp1-Cre line to knockout RXRα and Lrat, respectively, as we see in Trp1-Cre alone. Similarly, areas of hyperfluorescence that increased in intensity over time but not in size (window defect) on FA have been reported as a result of conditional ablation of vascular endothelial growth factor in the RPE.
      • Marneros A.G.
      • Fan J.
      • Yokoyama Y.
      • Gerber H.P.
      • Ferrara N.
      • Crouch R.K.
      • Olsen B.R.
      Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function.
      In addition, the mosaic nature of the Cre toxicity phenotype necessitates that as proper controls panoramic investigation of the RPE monolayer should be included in RPE conditional knockout studies. Therefore, on the basis of our findings in this study, we may need to re-evaluate the conclusions drawn from previous studies that used the Trp1-Cre mouse.
      • Jiang L.Q.
      • Jorquera M.
      • Streilein J.W.
      Subretinal space and vitreous cavity as immunologically privileged sites for retinal allografts.
      • Kanazawa H.
      • Ohsawa K.
      • Sasaki Y.
      • Kohsaka S.
      • Imai Y.
      Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma-dependent pathway.
      • Schmidt-Supprian M.
      • Rajewsky K.
      Vagaries of conditional gene targeting.
      • Lee J.Y.
      • Ristow M.
      • Lin X.
      • White M.F.
      • Magnuson M.A.
      • Hennighausen L.
      RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function.
      • Marneros A.G.
      • Fan J.
      • Yokoyama Y.
      • Gerber H.P.
      • Ferrara N.
      • Crouch R.K.
      • Olsen B.R.
      Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function.
      • Mori M.
      • Metzger D.
      • Picaud S.
      • Hindelang C.
      • Simonutti M.
      • Sahel J.
      • Chambon P.
      • Mark M.
      Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium.
      • Kobayashi T.
      • Urabe K.
      • Winder A.
      • Jimenez-Cervantes C.
      • Imokawa G.
      • Brewington T.
      • Solano F.
      • Garcia-Borron J.C.
      • Hearing V.J.
      Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis.
      Table 2Studies That Have Used the Trp1-Cre Mouse to Inactivate Targeted loxP Sites
      Targeted genePhenotype describedYearRef.
      RXRαDecreased expression of RPE65, CRALBP; photoreceptor alterations; decrease in number; shortening of OS; reduced light responses on ERG2004
      • Mori M.
      • Metzger D.
      • Picaud S.
      • Hindelang C.
      • Simonutti M.
      • Sahel J.
      • Chambon P.
      • Mark M.
      Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium.
      VEGFαMicrophthalmia; absence of choroid; reduction in the content of melanin granules; abnormally shaped melanin granules; disorganization of basal infoldings; loss of apical villi; reduced RPE thickness; ERG abnormalities (decrease in a and b wave); RPE defects on fluorescein angiography2005
      • Marneros A.G.
      • Fan J.
      • Yokoyama Y.
      • Gerber H.P.
      • Ferrara N.
      • Crouch R.K.
      • Olsen B.R.
      Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function.
      LratReduced light responses on ERG; shortening of rod outer segments; slight reduction in photoreceptor nuclei2007
      • Ruiz A.
      • Ghyselinck N.B.
      • Mata N.
      • Nusinowitz S.
      • Lloyd M.
      • Dennefeld C.
      • Chambon P.
      • Bok D.
      Somatic ablation of the Lrat gene in the mouse retinal pigment epithelium drastically reduces its retinoid storage.
      PTENRetinal degeneration; reduction in ONL; pigmented tumors in spleen2008
      • Kim J.W.
      • Kang K.H.
      • Burrola P.
      • Mak T.W.
      • Lemke G.
      Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium.
      CTNNB1 (β-catenin)Microphthalmia; colobomas; disruption of cellular junctions2009
      • Westenskow P.
      • Piccolo S.
      • Fuhrmann S.
      Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression.
      CTNNB1 (β-catenin)Microphthalmia; colobomas; disruption of cellular junctions2009
      • Fujimura N.
      • Taketo M.M.
      • Mori M.
      • Korinek V.
      • Kozmik Z.
      Spatial and temporal regulation of Wnt/beta-catenin signaling is essential for development of the retinal pigment epithelium.
      RBP-Jκ, Notch1, Notch2Microphthalmia; benign pigmented tumors2010
      • Schouwey K.
      • Aydin I.T.
      • Radtke F.
      • Beermann F.
      RBP-Jkappa-dependent Notch signaling enhances retinal pigment epithelial cell proliferation in transgenic mice.
      CRALBP, cellular retinaldehyde-binding protein; ONL, outer nuclear layer; OS, outer segment.
      Another interesting finding of this study is the activation of the retinal microglia and its subsequent migration to the subretinal space, which is normally devoid of immune cells.
      • Jiang L.Q.
      • Jorquera M.
      • Streilein J.W.
      Subretinal space and vitreous cavity as immunologically privileged sites for retinal allografts.
      The RPE plays an important role in keeping the retinal microglia in a ramified-quiescent state through the secretion of immunosuppressive cytokines such as transforming growth factor-β, IL-10, and pigment epithelium-derived factor among others.
      • Zamiri P.
      • Sugita S.
      • Streilein J.W.
      Immunosuppressive properties of the pigmented epithelial cells and the subretinal space.
      Indeed, IL-10 protein levels were found to be significantly decreased in choroid-RPE lysates from Trp1-Cre mice compared with wild-type controls (see Supplemental Figure S3N at http://ajp.amjpathol.org), which may partially explain the profound activation. However, it is not clear whether the reduction in IL-10 was because of a decrease in RPE cell number or because of a general manifestation of RPE dysfunction. In our study, the retinal microglia were strongly associated with areas of damaged RPE cells and phagocytosed cellular debris most likely originating from RPE cells. It is plausible that microglial cells play a role in repopulating and remodeling the RPE monolayer after dysfunction or cell death with the aim of maintaining its integrity. This is further supported by several recent studies showing that activated subretinal microglia can influence the function of RPE cells or even change their morphology.
      • Ma W.
      • Zhao L.
      • Fontainhas A.M.
      • Fariss R.N.
      • Wong W.T.
      Microglia in the mouse retina alter the structure and function of retinal pigmented epithelial cells: a potential cellular interaction relevant to AMD.
      • Xu H.
      • Chen M.
      • Manivannan A.
      • Lois N.
      • Forrester J.V.
      Age-dependent accumulation of lipofuscin in perivascular and subretinal microglia in experimental mice.
      The accumulation of activated subretinal microglia has been reported in specimens from patients with dry age-related macular degeneration.
      • Gupta N.
      • Brown K.E.
      • Milam A.H.
      Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration.
      Further investigations are needed to fully elucidate the interplay between the RPE and the retinal microglia.
      To date, six mouse lines expressing Cre recombinase in the RPE have been reported in the literature,
      • Mori M.
      • Metzger D.
      • Garnier J.M.
      • Chambon P.
      • Mark M.
      Site-specific somatic mutagenesis in the retinal pigment epithelium.
      • Le Y.Z.
      • Zheng W.
      • Rao P.C.
      • Zheng L.
      • Anderson R.E.
      • Esumi N.
      • Zack D.J.
      • Zhu M.
      Inducible expression of cre recombinase in the retinal pigmented epithelium.
      • Aydin I.T.
      • Beermann F.
      A mart-1: :Cre transgenic line induces recombination in melanocytes and RPE.
      • Iacovelli J.
      • Zhao C.
      • Wolkow N.
      • Veldman P.
      • Gollomp K.
      • Ojha P.
      • Lukinova N.
      • King A.
      • Feiner L.
      • Esumi N.
      • Zack D.J.
      • Pierce E.A.
      • Vollrath D.
      • Dunaief J.L.
      Generation of Cre transgenic mice with postnatal RPE-specific ocular expression.
      • Longbottom R.
      • Fruttiger M.
      • Douglas R.H.
      • Martinez-Barbera J.P.
      • Greenwood J.
      • Moss S.E.
      Genetic ablation of retinal pigment epithelial cells reveals the adaptive response of the epithelium and impact on photoreceptors.
      • Guyonneau L.
      • Rossier A.
      • Richard C.
      • Hummler E.
      • Beermann F.
      Expression of Cre recombinase in pigment cells.
      yet this is the first report to indicate potential problems caused by prolonged and robust Cre expression by the RPE. The possibility of such toxicity effect should alert investigators using other lines expressing Cre in the RPE. Although the use of transgenic animals with high levels of Cre expression can ensure a high recombination level, they may also pose a significant risk of Cre-mediated cellular toxicity potentially because of nonspecific “illegitimate” recombination events.
      • Loonstra A.
      • Vooijs M.
      • Beverloo H.B.
      • Allak B.A.
      • van Drunen E.
      • Kanaar R.
      • Berns A.
      • Jonkers J.
      Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells.
      To avoid this complication, several strategies have been developed to control Cre expression such as induction via tamoxifen or tetracycline administration or the use of self-deleting Cre-expressing vectors.
      • Pfeifer A.
      • Brandon E.P.
      • Kootstra N.
      • Gage F.H.
      • Verma I.M.
      Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.
      Despite these efforts, issues of toxicity remain even with ligand-dependent recombinases.
      • Hameyer D.
      • Loonstra A.
      • Eshkind L.
      • Schmitt S.
      • Antunes C.
      • Groen A.
      • Bindels E.
      • Jonkers J.
      • Krimpenfort P.
      • Meuwissen R.
      • Rijswijk L.
      • Bex A.
      • Berns A.
      • Bockamp E.
      Toxicity of ligand-dependent Cre recombinases and generation of a conditional Cre deleter mouse allowing mosaic recombination in peripheral tissues.
      • Forni P.E.
      • Scuoppo C.
      • Imayoshi I.
      • Taulli R.
      • Dastru W.
      • Sala V.
      • Betz U.A.
      • Muzzi P.
      • Martinuzzi D.
      • Vercelli A.E.
      • Kageyama R.
      • Ponzetto C.
      High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly.
      Therefore, it is clear that inclusion of the most crucial control, namely mice carrying the Cre transgene, and a comprehensive investigation of the RPE flat mount should always be included in conditional gene targeting studies for accurate interpretation of scientific results.

      Acknowledgments

      We thank Kip M. Connor and Patricia D'Amore for their useful advice, insightful discussions, and help during the preparation of this manuscript.

      Supplementary data

      • Supplemental Figure S1

        A: Genotyping for Cre-transgene (100 bp) and internal positive control (∼324 bp). B: PCR analysis for the presence of the retinal degeneration 1 mutation. Genomic DNA from FVB/N mice was used as positive control. C: X-Gal staining and subsequent melanin bleaching of Trp1-Cre; ROSA26R flat mounts shows variable degree of mosaicism in Cre expression. Scale bar = 500 μm. D: Whole mount X-Gal staining confirms ectopic Cre expression in the optic nerve stalk, as initially reported by Mori et al.

        • Mori M.
        • Metzger D.
        • Garnier J.M.
        • Chambon P.
        • Mark M.
        Site-specific somatic mutagenesis in the retinal pigment epithelium.

      • Supplemental Figure S2

        A: Double immunofluorescent staining of RPE flat mounts from Trp1-Cre mice and littermate wild-type controls with the use of antibodies against the proteins ZO-1 and β-catenin. Loss of the classic honeycomb appearance of the RPE monolayer can be observed. Scale bar = 200 μm. B: Immunofluorescent staining with an antibody against β-catenin on RPE flat mounts obtained at P14 indicates significant disorganization of the RPE monolayer. Scale bar = 100 μm.

      • Supplemental Figure S3

        Double immunofluorescent staining of RPE flat mounts using a FITC-conjugated antibody against ZO-1 and the microglial cell marker Iba-1. The subretinal space is devoid of microglial cells in wild-type mice (A–C), compared with Trp1-Cre mice in which significant microglial cell activation is observed. D–F: Microglial cell bodies co-localized strongly with cellular debris stained positive for FITC-ZO1. Scale bars: 100 μm (A–F). Microglial were often localized in areas of RPE remodeling. Notice the changes in the shape of RPE cells (arrows). Scale bar = 10 μm. Isotype control antibodies (K–M). Quantitative determination of IL-10 protein levels in choroid-RPE protein lysates obtained from Trp1-Cre and littermate controls (N).

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