December 2007
Volume 48, Issue 12
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Biochemistry and Molecular Biology  |   December 2007
Somatic Ablation of the Lrat Gene in the Mouse Retinal Pigment Epithelium Drastically Reduces Its Retinoid Storage
Author Affiliations
  • Alberto Ruiz
    From the Jules Stein Eye Institute, the
    Department of Neurobiology, and the
  • Norbert B. Ghyselinck
    IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire),
    INSERM (National Institute of Health and Medical Research), U596, CNRS (Centre National de la Recherche Scientifique), Illkirch, France;
    Université Louis Pasteur, Strasbourg, France; and
  • Nathan Mata
    Sirion Therapeutics, La Jolla, California.
  • Steven Nusinowitz
    From the Jules Stein Eye Institute, the
  • Marcia Lloyd
    From the Jules Stein Eye Institute, the
  • Christine Dennefeld
    IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire),
  • Pierre Chambon
    IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire),
    INSERM (National Institute of Health and Medical Research), U596, CNRS (Centre National de la Recherche Scientifique), Illkirch, France;
    Université Louis Pasteur, Strasbourg, France; and
  • Dean Bok
    From the Jules Stein Eye Institute, the
    Department of Neurobiology, and the
    Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, California;
Investigative Ophthalmology & Visual Science December 2007, Vol.48, 5377-5387. doi:10.1167/iovs.07-0673
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      Alberto Ruiz, Norbert B. Ghyselinck, Nathan Mata, Steven Nusinowitz, Marcia Lloyd, Christine Dennefeld, Pierre Chambon, Dean Bok; Somatic Ablation of the Lrat Gene in the Mouse Retinal Pigment Epithelium Drastically Reduces Its Retinoid Storage. Invest. Ophthalmol. Vis. Sci. 2007;48(12):5377-5387. doi: 10.1167/iovs.07-0673.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To generate a mouse model in which the Lrat gene is selectively disrupted in the retinal pigment epithelium (RPE). To evaluate the effects on the synthesis of retinyl esters and on the expression of other proteins involved in the continuation of the visual cycle.

methods. A mouse line in which part of the first exon of the Lrat gene has been flanked by loxP sites, was generated and used in the study (Lrat L3/L3 mice). Heterozygous mice (Lrat +/L3) were crossed with mice expressing Cre-recombinase under control of the tyrosinase-related protein-1 (Tyrp1) promoter, which is active selectively in melanin-synthesizing cells such as RPE cells. Accordingly, mice obtained from these crosses should display an RPE-specific disruption of the Lrat gene (Lrat rpe−/−). In addition, by crossing CMV-Cre transgenic mice with Lrat L3/L3 animals, a germline null Lrat knockout (Lrat L−/L− mice) was generated. RNA and protein expression, endogenous retinoid levels, and electroretinogram (ERG) analyses were performed on Lrat rpe−/− and Lrat L−/L− mice, to determine the effects of Lrat disruption. Retinoid levels in nonocular tissues were also analyzed for comparison.

results. Analysis of RPE tissues from Lrat rpe−/− mice showed absence of Lrat message, lack of Lrat protein expression and consequently a reduced light response in ERG recordings. In addition, RPE cells from Lrat rpe−/− showed a strong reduction in their ability to synthesize all-trans retinyl esters, whereas Lrat activity in other tissues known to process retinol was comparable to control Lrat L3/L3 animals. The Lrat L−/L− mice showed no detectable Lrat message, lack of protein expression, and barely detectable ester formation in RPE cells or several other relevant tissues analyzed.

conclusions. Three Lrat mouse lines with genetic modifications were generated. The Lrat L−/L− mice displayed features similar to equivalent models previously reported by others. The second mouse line (Lrat rpe−/−) displayed loss of Lrat function only in the RPE. The third line possesses functional Lrat in all tissues, but part of the Lrat coding gene was flanked by loxP sites (Lrat L3/L3). This feature allows the disruption of this gene in any tissue of choice, by intercrossing with mice in which Cre-recombinase expression is driven by an appropriate tissue-specific promoter.

The retinal pigment epithelium (RPE) of the eye is a multifunctional component of the vertebrate retina. 1 Anatomically, this epithelium is composed of a monolayer of cuboidal cells and is located between the large-bore capillary bed of the choroidal layer and the rod and cone photoreceptors of the neurosensory retina. One of the most important functions of the RPE in vision is the uptake of vitamin A (retinol) and the processing and transport of its derivatives (retinoids) in a cascade of enzymatic reactions known as the visual cycle. 1 2 3  
In this cycle, retinol is initially delivered to the RPE cells from the liver as a complex with serum retinol-binding protein (RBP) and transthyretin. 4 This complex is bound to the basolateral plasma membrane of RPE cells via interaction with an RBP receptor. 5 Recently, a multitransmembrane protein of previously unknown function, stimulated by retinoic acid 6 (STRA6) has been suggested as a strong candidate for the RBP receptor. 6 This receptor binds RBP and delivers retinol into the cell where it is bound by cellular retinol binding protein 1 (CRBP1) for presentation to lecithin retinol acyltransferase (LRAT). 7 LRAT transfers fatty acyl groups from the sn-1 position of phosphatidylcholine (lecithin) to all-trans retinol for the generation of the all-trans retinyl esters. 8 9 All-trans retinyl esters are converted to 11-cis retinol via an isomerization reaction in which the RPE65 protein plays a critical role. 10 11 12 11-cis Retinol is subsequently bound to cellular retinaldehyde-binding protein (CRALBP), oxidized into 11-cis retinaldehyde by 11-cis retinol dehydrogenase (11c-RDH) and then transported to the plasma membrane for its apical release from the RPE. 13 14 Finally, 11-cis retinaldehyde is exported to photoreceptors where it combines with opsin for the formation of rhodopsin and cone photopigments. 
In addition to its important role in the continuation of the visual cycle, LRAT is also critical in retinol processing in several other tissues, such as liver, 15 testis, 16 and small intestine. 8 Therefore, defects in this gene are predicted to have adverse effects on vision and retinoid metabolism. In humans, null mutations of visual cycle genes such as, RPE65, RDH5 (encoding 11c-RDH), RLBP1 (encoding CRALBP), and RGR (retinal-G-protein coupled receptor) have been related to inherited retinal degenerations. 17 18 19 20 21 22 In the case of LRAT, mutations in this gene have been associated with early-onset severe retinal dystrophy, the phenotype Leber congenital amaurosis (LCA). 23 To understand more about the adverse impact on the visual cycle process caused by disruption of the Lrat gene, the generation of a knockout mouse model would be useful. Currently, two different groups have developed mouse lines bearing a germline null mutation of Lrat, and have highlighted the importance of these mutants in the study of human retinal dystrophies and as a model for vitamin A deprivation. 24 25 However, because of the importance of Lrat in essential processes such as reproduction, immune function, and development, 26 the maintenance and propagation of such mouse lines represents a limitation—mainly due to limited male fertility. 24 25 Using the Cre/loxP technology, 27 28 we generated a mouse line in which the Lrat gene was selectively disrupted in the RPE of the eye. We demonstrate that, in addition to a drastic reduction of intracellular retinyl esters and impaired vision, selective disruption of the Lrat gene in the RPE also affects the expression of Crbp1, Rpe65, and RGR. These data confirm that Lrat is critical for the generation of all-trans retinyl esters and 11-cis retinoids and suggests its possible regulatory role on the expression of other proteins involved in the visual cycle. 
Materials and Methods
Targeting Vector and Homologous Recombination
A 129/SvJ mouse genomic library constructed in a vector (Lambda Fix II; Stratagene, La Jolla, CA) was screened with a gel-purified 697-bp PCR product containing the entire Lrat coding sequence. The Lrat coding sequence was amplified from RNA PolyA+ extracted from mouse liver using primers 5′-atgaagaacccaatgctgga-3′ and 5′-ctagccagacatcatccaca-3′. Two phages containing ∼14.8- and ∼12.9-kb genomic inserts, respectively, were isolated. The ∼12.9-kb fragment spanned the entire Lrat gene and was used for the construction of the targeting vector. A loxP site (lowercase sequence) was created by inserting a pair of complementary oligonucleotides 5′-AGACGTCataacttcgtataatgtatgctatacgaagttatCAGCT-3′ and 5′-GataacttcgtatagcatacattatacgaagttatGACGTCTAGCT-3′ into an SacI site located in exon 1, 49 nucleotides upstream of the ATG initiation codon. A NotI site was created by inserting a second pair of complementary oligonucleotides (5′-GATCAGCGGCCGCCATATGGATATCG-3′ and 5′-CTAGGGATATCCATATGGCGGCCGCT-3′) into the BamHI site located in the single intron, 35 nucleotides downstream from exon 1. A ∼1.7-kb NotI-restricted fragment containing a loxP-flanked neomycin-resistance gene (PGK-NeoA+, gift from Daniel Metzger, IGBMC; Institut de Génétique et de Biologie Moléculaire et Cellulaire) was then inserted into the created NotI site of the Lrat gene. The resultant ∼15-kb HpaI-digested purified targeting vector was electroporated into P1 embryonic stem (ES) cells as described. 29 After selection with G418, 372 resistant clones were expanded. Genomic DNA was prepared from each clone, restricted with EcoRV and analyzed by Southern blot using a 5′ probe external to the targeting vector, which consisted of a 596-bp PCR fragment located ∼4-kb upstream of the ATG. An 8.4-kb fragment was obtained for the wild-type (WT) allele, whereas a 6.0-kb fragment was obtained for the targeted (L3) allele. Genomic DNA was also restricted with SacI, and analyzed by Southern blot using a 3′ probe external to the targeting vector, which consisted of an 807-bp PCR fragment located ∼4.7 kb downstream from the ATG. A 5.6-kb fragment was obtained for the WT allele, whereas a 8.0-kb fragment was obtained for the targeted allele (data not shown). One correctly targeted ES cell clone (K100-36) was injected into C57BL/6 blastocysts, and male chimeras derived from it gave germline transmission, thereby generating Lrat +/L3 mice. 
Animals and Genotyping
All experimental procedures were performed on 2- to 5-month-old mice (unless otherwise indicated), which were on a C57BL/6J background. All mice were maintained in a normal 12 hours on and 12 hours off light cycle and fed ad libitum with NIH-31 modified open formula, which contains 24,500 IU/kg vitamin A acetate (Harlan Teklad, Madison, WI). All experiments involving the use of mice were in compliance with the guidelines established by the National Institutes of Health and by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Genotyping was performed by PCR amplification of genomic DNA from a tail biopsy using a combination of the primer sets described in Table 1 . The presence of the Tyrp1-Cre transgene was assessed using primers 5′-atttgcctgcattaccggtc-3′ and 5′-atcaacgttttcttttcgga-3′, which allow the amplification of a 350-bp PCR fragment. 30 Note that the founder mice harboring the Tyrp1-Cre transgene were genotyped for the rd (retinal degeneration) mutation 31 that is present in the original FVB/N background, 32 and only mice that did not carry the rd mutation were used for breeding with Lrat L3/L3 mice. Also, to avoid an indirect effect on animal variability due to their rpe65 genotype, only mice homozygous for the Leu450 allele were used. 
Preparation of RPE Cells
RPE cells were collected from freshly dissected eyes under a stereomicroscope. The anterior segment, lens, and retina were first removed. The eye cup was then rinsed in calcium- and magnesium-free Hanks’ balanced salt solution (HBSS). Sheets of RPE cells were carefully peeled away from their choroidal attachment with fine forceps. Cells thus collected were transferred with a dropper pipette into fresh HBSS on ice. RPE cells were used immediately or temporarily stored at −80°C. 
Western Blot Analysis
Microsomal proteins from several freshly dissected tissues including RPE, brain, heart, liver, lung, kidney, testis, and pancreas were extracted as described before. 33 All protein measurements were performed in a spectrophotometer (model ND-1000; NanoDrop Technologies Inc., Wilmington, DE). Protein (10 μg) from each microsomal sample was used for analysis. Native bovine RPE cells were included as positive controls for Lrat detection. Samples were boiled for 2 minutes before electrophoresis by 12.5% (wt/vol) SDS-PAGE (Bio-Rad, Hercules, CA). Proteins were transferred to nitrocellulose membrane filters (Schleicher−Schuell, Sacramento, CA). Each filter membrane was separately incubated for 1 hour at room temperature with antibodies described in Table 2 . Monoclonal and polyclonal antibodies were used at dilutions of 1:5000 and 1:1000, respectively. The specificity of these antibodies has been shown. 24 34 35 36 37 38 39 Protein bands were visualized by using the enhanced chemiluminescence (ECL) Western blot system (Amersham, Arlington Heights, IL). The stripping process for reprobing was accomplished by incubating the filter membranes in 0.2 M glycine (pH 2.8), at room temperature for 30 minutes followed by two, 10 minutes washes. 
Immunohistochemistry
For detection of Lrat on RPE sections, eyes were fixed by intracardiac perfusion 40 of ice-cold 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS; 136 mM NaCl, 11.4 mM sodium phosphate [pH 7.4]), then kept in the same fixative for 4 hours at 4°C, washed in PBS, and frozen in OCT embedding medium. Sections (20-μm-thick) were first incubated for 30 minutes at room temperature in 20% (vol/vol) normal goat serum diluted in PBS-T buffer [PBS containing 0.1% (vol/vol) Tween-20], then incubated for 2 hours at room temperature with the Lrat mouse monoclonal antibody 24 diluted at 1:50 in PBS-T and containing 20% (vol/vol) normal goat serum. Detection of bound primary antibodies was achieved by incubating the sections for 1 hour at room temperature, using a Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:500. Nuclei were counterstained with 4′, 6-diamidino-2-phenyl-indole (DAPI; Roche Diagnostics, Meylan, France) diluted in the mounting medium at 10 μg/mL (Vectashield; Vector Laboratories, Burlingame, CA). Sections were imaged with a confocal microscope (ST1; Leica, Wetzlar, Germany). 
Light Microscopy
Mice anesthetized with carbon dioxide were fixed by intracardiac perfusion with a mixture of 2% (wt/vol) paraformaldehyde and 2.5% (wt/vol) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2). Light cautery was applied at the superior pole of the cornea to mark the orientation before enucleation. A window was cut in the cornea, and the eye was immersed in primary fixative and rotated at room temperature for 2 hours. The anterior segment was removed, and the remaining eye cup was refrigerated overnight in primary fixative. The eye cup was trimmed into temporal and nasal hemispheres. The tissues were immersed in 1% (wt/vol) osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.2) for 1 hour followed by dehydration in a graded series of alcohols. The temporal hemispheres were embedded in an Epon/Araldite mixture (25 parts/15 parts). Light microscope sections were cut at 1 μm and stained with 1% (wt/vol) toluidine blue and 1% (wt/vol) sodium borate, then photographed with a light microscope (63× oil lens) (LSM 210; Carl Zeiss Meditec, Dublin, CA) equipped with a digital camera (Coolsnap; Roper Scientific, Duluth, GA). 
ERG Analysis
Mice, dark-adapted overnight, were anesthetized with an intraperitoneal injection of normal saline containing ketamine (15 mg/g) and xylazine (3 mg/g body weight). ERGs were recorded from the corneal surface of one eye after pupil dilation 1% [wt/vol] atropine sulfate in saline buffer) using a gold-loop electrode referenced to a similar gold wire in the mouth. All stimuli were generated with a photic stimulator (model PS33 Plus; Grass-Telefactor, West Warwick, RI) affixed to the outside of the dome at 90° to the viewing porthole. ERGs were recorded to a series of blue (Wratten 47 A; λ max = 470 nm) flashes of increasing intensity, ranging from −4.7 to −0.44 log cd-s/m2. Responses were sampled at 1 kHz, amplified (CP511 AC amplifier, ×10,000; Grass-Telefactor) and digitized using an I/O board (PCI-1200; National Instruments, Austin, TX) in a personal computer. Signal processing was performed with custom-written software. For each stimulus, responses were computer averaged with up to 50 records averaged for the weakest signals. A signal rejection window could be adjusted online to eliminate artifacts. 
Retinoid Analysis
All procedures were performed under dim red light. The mice were killed, and samples of RPE (posterior eyecup minus retina), liver, lung, testis, and kidney were flash frozen and stored at −80°C. At time of analysis, the tissue samples (0.15–0.75 g wet weight) were thawed in 1 to 2 mL PBS (pH 7.2) and homogenized with a tissue grinder (PowerGen; Fisher Scientific, Houston, TX). Retinoids in the sample extract were analyzed by normal phase high-performance liquid chromatography, as previously described. 33  
RT-PCR Analysis
RPE cells and other tissues including brain, heart, liver, lung, kidney, testis, and pancreas were collected from mice with the indicated genotypes. Total RNA was extracted (RNeasy; Qiagen, Valencia, CA). Reverse transcription coupled to polymerase chain reaction (RT-PCR) experiments were performed with an RNA-PCR kit, according to the manufacturer’s recommendations (Applied Biosystems [ABI], Branchburg, NJ). Primer sets specific for each gene are described in Table 3 . All experiments were performed with a temperature cycler (Robocycler 40; Stratagene, La Jolla, CA). To check for identity, PCR products were cloned into a vector (pCR II; Invitrogen, San Diego, CA), and sequenced with dye termination chemistry (DYEnamic ET Terminator Kit; GE Healthcare, Piscataway, NJ) in a genetic analyzer (Prism 3100 Genetic Analyzer; ABI). 
Results
Generation of Mice Harboring a Conditional LoxP-flanked Lrat allele
To create mice in which the Lrat gene can be selectively ablated in the RPE or in any other tissue of interest, a loxP-flanked Lrat allele was engineered in ES cells through homologous recombination (Fig. 1A) . The ES cell clone K100-36, harboring the conditional (L3) Lrat allele, was used to generate a mouse line (Lrat +/L3 mice). By using primers P1 and P2 (Table 1) , a 376-bp PCR product was obtained for the WT allele, whereas a 423-bp fragment was obtained from the L3 allele, thereby allowing discrimination of heterozygous and homozygous progenies from Lrat +/L3 intercrosses (Fig. 1B) . In addition, PCR amplification of a 741-bp fragment using primers P5 and P6 consistently confirmed the presence of the loxP-flanked PGK-NeoA+ selection cassette in mice harboring the L3 allele (data not shown). Heterozygous matings (n = 12, mean of seven pups per litter) yielded 27% (n = 23) WT (Lrat +/+), 49% (n = 41) heterozygous (Lrat +/L3), and 24% (n = 20) homozygous (Lrat L3/L3) mice. The latter were healthy, fertile, and indistinguishable from WT littermates. Lrat mRNA and Lrat protein levels were normal in Lrat L3/L3 mouse tissues such as RPE and liver (data not shown), indicating that neither the presence of loxP sites nor PGK-NeoA+ in the genome of Lrat L3/L3 mice affects the expression of Lrat protein (see Figs. 2 7 ). 
To check whether Cre-mediated excision at the Lrat locus can occur in vivo, Lrat L3/L3 mice were crossed with CMV-Cre transgenic mice, which express Cre recombinase in germ cells. 28 The resultant mice (Lrat +/L−) transmitting the excised allele (L−) in their germline were identified by Southern blot (data not shown). Identification of heterozygous and homozygous progenies from Lrat +/L− intercrosses was performed by PCR analysis of tail DNA. By using primers P3 and P4 (see Table 1 ), a 346-bp PCR product was obtained for the L− allele, whereas an 877-bp fragment was obtained from the WT allele (Fig. 1C) . Heterozygous mating (n = 32, mean of six pups per litter) yielded 25% (n = 48) WT (Lrat +/+), 52% (n = 101) heterozygous (Lrat +/L−), and 23% (n = 43) homozygous (Lrat L−/L−) mice. The latter were externally indistinguishable from WT littermates. To verify that excision generated a null allele, we determined whether Lrat protein could be detected in RPE tissues of Lrat L−/L− mice. We performed Western blot analysis on microsomal proteins from WT and Lrat L−/L− RPE tissues using an Lrat monoclonal antibody 24 (Fig. 1D , top). A protein band with a molecular mass of ∼25 kDa was detected in RPE cells of WT mice and was comparable to the one observed in bovine RPE cells used as a positive control for Lrat detection. In contrast, no Lrat protein was detected in RPE extracts from Lrat L−/L−mice. A smaller protein of ∼23 kDa was also observed in WT extracts, the identity of which is currently unknown. This protein is probably related to Lrat, as it is not detected in the RPE extracts of mutant mice. The expression of actin (∼43 kDa) was subsequently determined to monitor protein loading among the samples (Fig. 1D , bottom). To demonstrate further that Lrat gene disruption is efficient, we used immunohistochemistry (Fig. 1E) . When compared with a WT littermate’s eye (Fig. 1E , left), no staining for Lrat was detected in the eye of Lrat L−/L− adult mice (Fig. 1E , right), indicating that these mutants lack Lrat. Thus, Cre-mediated recombination of the Lrat L3 allele into L− actually produced a null allele. 
Somatic Ablation of Lrat in Mouse RPE Tissue
To selectively inactivate the Lrat gene in the RPE, we used mice carrying a transgene made of the Cre recombinase-coding sequence placed under control of the tyrosinase-related protein-1 (Tyrp1) promoter, 41 which is active in melanin-synthesizing cells and therefore drives gene expression in the RPE. 42 This Tyrp1-Cre transgene is active in the RPE from embryonic day 10.5 to postnatal day 12, resulting in excision of any loxP-flanked DNA segment in the RPE from embryonic day 10.5 to adulthood. 42 Thus, Lrat L3/L3 females were crossed with males bearing both the transgene and one L3 allele of Lrat (i.e., Typr1-Cre tg/0/Lrat +/L3 mice). These crosses generated control (i.e., Lrat L3/L3, also designated as WT) and experimental (i.e., Tyrp1-Cre tg/0/Lrat L3/L3) mice. No Lrat protein was evidenced in RPE from Tyrp1-Cre tg/0/Lrat L3/L3 animals, which were therefore referred to as Lrat rpe−/− mutants (Fig. 1D , top). a protein band corresponding to Lrat was detected in other tissues of Lrat rpe−/− mice known to process retinol including liver, testis, lung, and kidney, as well as in others tissues such as brain, heart, and pancreas (Fig. 2 , bottom). This observation indicates that somatic ablation of Lrat actually occurred selectively in RPE. In comparison, Lrat protein could not be detected in tissues from Lrat L−/L− mice, as expected for a germline null mutation (Fig. 2 , middle). RT-PCR experiments performed on RNA extracted from several tissues of Lrat rpe−/− mice confirmed that Lrat gene ablation occurred selectively in RPE tissues (data not shown). 
PCR Analysis of Lrat Cre-Mediated Excision in Tissues from Lratrpe−/− Mice
Genomic DNA was extracted from RPE, brain, heart, liver, lung, testis, and pancreas of Lrat rpe−/− mice in an attempt to detect Lrat Cre-mediated excision in tissues other than RPE. As shown in Figure 3 , a 423-bp band (L3 allele) was observed in all tissues including RPE of Lrat L3/L3 controls, using the P1-P2 primer set described in Table 1(Fig. 3 , first panel). Of note, this band was absent in the RPE of Lrat rpe−/− mice after PCR amplification with the same set of primers (Fig. 3 , second panel). In contrast, only the RPE and no other tissue from Lrat rpe−/− mice showed a 346-bp band (L− allele) when using the P3-P4 primer set (Fig. 3 , third panel). This 346-bp band was observed in all tissues, including the RPE of Lrat L−/L− mice used as a reference in this analysis (Fig. 3 , fourth panel). 
Effect of Somatic Ablation of Lrat in RPE on the Histologic Appearance of the Retina Outer Segment and Nuclear Layer
Comparison of retina sections from WT (Lrat +/+) and Lrat L3/L3 control animals with sections from Lrat L−/L− and Lrat rpe−/− mutants at 6 and 8 weeks and at 6 months of age revealed a pronounced shortening in rod outer segment length and a slight reduction of the number the photoreceptor nuclei (Fig. 4) . These observations suggest the importance of all-trans retinyl esters generated by Lrat in the RPE under normal conditions, and the adverse consequences on the outer segments due to the absence of Lrat in RPE cells. 
Effect of Somatic Ablation of Lrat in the RPE on the ERG Response
Representative dark-adapted ERG recordings for WT, Lrat L3/L3, Lrat L−/L−, and Lrat rpe−/− mice are shown in Figure 5 . Figures 5A 5B 5C 5Dshow the average response to progressively higher intensities, as indicated for the WT mouse in Figure 5A . The responses for the Lrat L3/L3 mouse (Fig. 5B)were well formed and were of the same order of magnitude as those from the WT mouse (Fig. 5A) . The saturated b-wave amplitude, obtained from a Naka-Rushton fit, was 558.0 and 630.0 μV for the WT and Lrat L3/L3 mice, respectively. ERG responses were nondetectable for the Lrat L−/L− mouse (Fig. 5C) , as previously described for another Lrat mutant line. 24 These data further confirmed that the Cre-mediated recombination of the Lrat L3 allele into L− actually produced an Lrat loss of function. In contrast, a small residual response was observed for the Lrat rpe−/− mouse (Fig. 5D , asterisk), but only at the brightest stimulus intensities. To confirm the existence of this small signal, ERGs were recorded to a 10-Hz flash stimuli (blue, −0.87 log cd-s/m2) under dark-adapted conditions with significant signal averaging. Representative recordings for WT, Lrat L−/L−, and Lrat rpe−/− mice are shown in Figure 5E . A recordable signal was observed in the Lrat rpe−/− mice that was not present in the Lrat L−/L− mice. The response waveforms were analyzed by fast Fourier transform (FFT), to derive estimates of signal and noise at the fundamental stimulus frequency (10 Hz). The FFT amplitude for the Lrat rpe−/− mouse in Figure 5Eexceeded that of noise (defined as the amplitude at twice the fundamental frequency) but did not for the Lrat L−/L− mouse, thereby confirming the existence of a very small residual response in the Lrat rpe−/− line. 
Effect of Somatic Ablation of Lrat in RPE on Retinoid Storage in the Eye Cup
The levels of endogenous retinoids in tissues known to express Lrat—namely, RPE, liver, lung, testis, and kidney—were evaluated (Table 4)as a surrogate measure of Lrat activity. In agreement with studies previously described, 24 25 all-trans retinyl esters were barely detectable in nonocular tissue samples from Lrat L−/L− mice. In contrast, nonocular tissue samples from Lrat rpe−/− contained levels of all-trans retinyl esters that were comparable to its control Lrat L3/L3 and to WT (Lrat +/+) control mice, except for liver and lung which showed smaller amounts. Of note, both Lrat L−/L− and Lrat rpe−/− mice contained very low, but detectable, levels of all-trans retinyl esters in the RPE (∼9% and ∼18%, respectively, of the amount measured in corresponding control mice), indicating that some residual retinol esterification activity may exist in RPE of both mutant mice (Table 4 ; Fig. 6 ). 
Effect of Somatic Ablation of Lrat on the Expression of Other Visual Cycle Proteins in RPE
To analyze the expression of genes whose products are involved in the visual cycle, we performed RT-PCR analysis on total RNA samples extracted from RPE tissues of control and Lrat mutant mice. We additionally included the genes coding for acyl coenzyme-A, diacylglycerol acyltransferase-1 (Dgat1), and Stra6, because it has recently been demonstrated that STRA6 is the long sought RBP receptor 6 and that DGAT1 could contribute to retinol esterification through an acyl-CoA-dependent reaction in the absence of LRAT. 43 The set of primers used during this analysis are described in Table 3 . As shown in Figure 7 , the Rpe65, Rdh5, Rlbp1, Rgr, Rbp1, Dgat1, and Stra6 mRNAs coding for Rpe65, 11c-Rdh, Cralbp, Rgr, Crbp1, Dgat1, and Stra6, respectively, were amplified both from WT, Lrat L−/L−, and Lrat rpe−/− RPE tissues. Despite the absence of Lrat transcripts in the mutants (Fig. 7 , lane 2), only slight variations in the amount of amplified products were observed for these genes. 
To further analyze their expression at the protein level, we performed Western blot analysis on RPE microsomal extracts of control and mutant mice by using the set of polyclonal and monoclonal antibodies described in Table 2 . No difference was observed in the expression of Stra6 protein in RPE samples from Lrat L−/L− and Lrat rpe−/− mutant mice, compared with the respective WT (Lrat +/+) and Lrat L3/L3 control (Fig. 8 , top, lanes 1–4). Because it is known that STRA6 is normally expressed in testis, 38 we used this tissue as reference to determine whether any variation among the Lrat mutants and control animals existed. Of note, the pattern of expression seen in RPE samples was also observed in protein extracts from testis of the same set of animals (Fig. 8 , top, lanes 5–8). Similarly, no variation was observed among Lrat mutants and control animals after RPE samples were reacted against Dgat1 antibodies (Fig. 8 , bottom, lanes 1–4). In this case we used Wat (white adipose tissue) as a reference, since WAT has been reported to express Dgat1 under normal conditions. 44 Our results showed that Dgat1 is equally expressed among Wat samples of Lrat mutants and controls and was highly expressed compared with RPE tissues (Fig. 8 , bottom, lanes 5–8). The analysis of visual cycle proteins is shown in Figure 9A . The expression of Crbp1 protein was two- to threefold reduced in the RPE of Lrat rpe−/− mice (Fig. 9 , compare lane 3 with lane 4). Other proteins such as Rpe65, as well as Rgr, which appears to modulate isomerohydrolase activity, 45 were highly attenuated in the RPE of Lrat rpe−/− mice (Fig. 9 , compare lane 5 with lane 6, and lane 7 with lane 8, respectively). In contrast, no variation of expression was found in the case of Cralbp (Fig. 9 , compare lane 9 with lane 10). Subsequent detection with an anti-actin monoclonal antibody showed that loading of the proteins was similar in each lane (Fig. 9B) . Detection with the anti-Lrat antibody showed that Lrat rpe−/− mutants lack Lrat in RPE tissue. Taken together, our data therefore demonstrate that ablation of Lrat in RPE alters the steady state level of other proteins involved in the visual cycle. 
Discussion
Lrat catalyzes an enzymatic reaction that transforms dietary vitamin A (all-trans retinol) into a stable retinyl ester storage form. Lrat is present in many tissues, including liver, testis, lung, small intestine, and the RPE, where it plays a key role in retinol metabolism. The mRNA from human Lrat was initially cloned and shown to encode for an integral membrane protein of 230 residues. 46 This finding facilitated the cloning of Lrat in other species such as rat and mouse. 47 In the eye, all-trans retinyl esters serve as the substrate for the isomerase of the visual cycle. The role of the isomerase is to generate 11-cis retinol, which is the immediate precursor of the visual chromophore used in rod and cone photoreceptors. 48 To examine the consequences of Lrat deficiency in the visual cycle, we generated a mouse line with selective inactivation of the Lrat gene in the RPE (Lrat rpe−/−). For comparison, we have also generated a mouse line with germline disruption of Lrat (i.e., Lrat L−/L− mutants). Neither of these mutants expresses Lrat mRNA or Lrat protein in the RPE. Consequently, both lines demonstrate a pronounced dark-adapted ERG deficit compared to control mice (Lrat L3/L3 and WT). As expected from previous studies, 24 Lrat L−/L− mice showed no detectable EGR response at the brightest stimuli. More important, ERG recordings for Lrat rpe−/− mice had intensity responses of 15 to 20 μV, which represents less than 5% of the 630 μV for the Lrat L3/L3 control subjects. These observations are consistent with the morphologic differences between retina sections from Lrat mutant and control mice (Fig. 4) . We conclude therefore that ablation of Lrat selectively in RPE cells is sufficient to compromise severely the response to light. 
RPE cells from Lrat rpe−/− mice showed a notable reduction in the expression of proteins that have close interaction with Lrat during the visual cycle process. Based on the intensity of the actin internal control in each set of samples, Crbp1 and Rpe65 had a reduction of approximately two- to threefold compared with the control, whereas in the case of Rgr, the reduction was even higher, approximately five- to sixfold. Because it is now known that RPE65, RGR and CRALBP are all involved in the regulation of isomerization activity, 10 11 12 45 49 it is tempting to hypothesize that these proteins function as a complex to generate and mobilize the visual chromophore. The altered expression or the absence of one of these proteins may have an effect on the stability of the others within the complex. Based on our observations, we can speculate that, under normal conditions, Lrat could play a regulatory role in the recruitment of partner proteins, and that absence of Lrat affects their steady state levels (or their stability). 
Quantification of endogenous all-trans retinyl esters was used as a surrogate measure of Lrat activity. Small amounts of all-trans retinyl esters were detected in RPE extracts of Lrat rpe−/− mice. This finding could be related to a partial excision of the loxP-flanked Lrat fragment by the Cre recombinase in the RPE. However, the Tyrp1-Cre transgenic line used in the present study has been shown to efficiently remove the loxP-flanked retinoid X receptor alpha (Rxra) gene in mouse RPE. 41 50 In addition, nondetectable levels of both Lrat mRNA and Lrat protein in the RPE of Lrat rpe−/− mice argues against the possibility of an incomplete gene ablation (Figs. 1D 1E 7 9) . In contrast, Lrat Cre-mediated excision in Lrat rpe−/− mice occurred selectively in the RPE and not in other tissues analyzed in Figure 3 . Along these lines, a residual amount of all-trans retinyl esters is also detected in the RPE of Lrat L−/L− mice, in which the two Lrat alleles have been deleted in the germline (i.e., a situation in which partial excision is not possible). We reasoned therefore that an incomplete Lrat loss of function in the RPE of Lrat rpe−/− mice could be ruled out. However, the esters detected in Lrat rpe−/− (and Lrat L−/L−) RPE may result from an alternate retinyl ester synthase activity like acyl CoA-retinol acyltransferase (Arat). 51 52 Although the acyl group transferred to retinol in this reaction comes from palmitoyl-CoA, the esters generated by Arat, as those generated by Lrat, maintain an all-trans stereochemical configuration. Therefore, the possibility exists that an ARAT activity accounts for the presence of all-trans retinyl ester in Lrat rpe−/− extracts. However, the retinyl esters levels in the RPE of Lrat rpe−/− (and Lrat L−/L−) mice represent only ∼18% (and ∼9%) of the levels in control mice, which is significantly below the 50% expected, when considering that at least 50% of the retinyl ester synthase activity in bovine RPE should be due to Arat. 52 In addition, it has been suggested that Arat activity in bovine RPE 52 and rat liver 53 will be triggered only in cases when concentrations of retinol are high. The fact that retinyl ester concentrations in Lrat rpe−/− and Lrat L−/L− mice are well below wild-type levels suggests that intracellular retinol would be equally low. Therefore, the retinyl esters present in the RPE of these mice probably cannot be attributed to Arat activity. 
Another pathway for generating retinyl esters is through the activity of DGAT1, which has recently been demonstrated to display an ARAT-like activity in vitro. 54 This enzyme is expressed in intestine, mammary gland, liver, and mainly in WAT. 44 Although the enzyme has not been reported in the RPE, we were able to amplify RNA message coding for Dgat1 and detect its protein expression at similar levels in RPE samples from Lrat rpe−/− and Lrat L−/L− and control mice (Figs. 7 8) . Although it appears that Dgat1 has a greater affinity for monounsaturated oleoyl-CoA over the saturated palmitoyl-CoA and all-trans retinol for the generation of retinyl esters, 54 55 it is conceivable that this enzyme could be responsible for the all-trans retinyl esters present in Lrat rpe−/− and Lrat L−/L− RPE. The generation of a double-mutant Lrat L−/L−/Dgat −/− would help resolve this issue. In either case, the possibility of ester formation by a yet undiscovered enzyme should not be discarded. 
The most parsimonious explanation at this time could be that, because all-trans retinyl esters are still normally produced by several other tissues in these Lrat rpe−/− mice (Table 4) , the esters detected in RPE extracts could be coming from the blood. It is known that retinyl esters processed in the small intestinal epithelium from retinol absorbed from the diet can be incorporated into the hydrophobic core of chylomicrons. The latter are then secreted into the lymph and ultimately enter the blood circulation via the thoracic and other lymphatic ducts. 56 The chylomicrons could then be delivered to the basolateral surface of the RPE, which has detectable levels of LDL receptors for its incorporation. According to this scenario, our data demonstrate that ∼90% of the retinyl esters present in RPE arise from RPE cell autonomous de novo synthesis, whereas ∼10% come from the blood circulation. 
In summary, we have generated three mouse lines that can be useful for the study of retinal dystrophies and retinol metabolism. The first one harbors a germline null mutation of Lrat gene, and the corresponding Lrat L−/L− mice display features similar to those previously reported for equivalent mouse models. 24 25 The second one, bearing an Lrat loss of function only in the RPE (Lrat rpe−/− mice), represents an important model for the study of Lrat’s impact, not only as an Acyl-transferase but also as a palmitoyl-transferase, as has been suggested recently. 57 Finally, mice from the third line we generated conserve a functional Lrat in all tissues, but part of their Lrat encoding gene has been flanked by loxP sites (Lrat L3/L3 mice). This genetic modification provides a versatile model to achieve the selective inactivation of Lrat in other tissues through the additional use of various transgenic mice in which Cre-recombinase expression is controlled by a tissue-specific promoter. 
 
Table 1.
 
Sequence of Primers Used for Genotype Analysis
Table 1.
 
Sequence of Primers Used for Genotype Analysis
Name Sequence Gene Expected Size of Allele (bp)
WT(+) L3 L−
P1 5′-aggagaagtcagggtgcaga-3′ Lrat 376 423 NA
P2 5′-tcatcctaaggcaaccaacc-3′
P3 5′-gcactttggcttctcctctg-3′ Lrat 877 346
P4 5′-gtggccacacccttttattg-3′
P5 5′-ggttctccggccgcttgggt-3′ PGK-NeoA+ NA* 741 NA
P6 5′-gaaggcgatgcgctgcgaat-3′
Table 2.
 
Antibodies Used for Western Blot Analysis
Table 2.
 
Antibodies Used for Western Blot Analysis
Protein Calculated MW (kDa) Antibodies Antigen Origin Source Reference
ACTIN 43 Monoclonal Rat Lin 36
CRALBP 36 Polyclonal Bovine Bok Unpublished (1984)
CRBP1 16 Polyclonal Rat Bok et al. 35
DGAT1 57 Polyclonal Mouse Chen et al. 39
LRAT 25 Monoclonal Mouse Batten et al. 24
RGR 32 Polyclonal Mouse Tao et al. 34
RPE65 61 Polyclonal Bovine Redmond & Hamel 37
STRA6 74 Polyclonal Mouse Bouillet et al. 38
Table 3.
 
Sequence of Primers Used for RT-PCR Analysis
Table 3.
 
Sequence of Primers Used for RT-PCR Analysis
Gene Protein Accession Number Sequence Position Size (bp)
Dgat1 DGAT1 AF078752 5′-gcgtctcttaaagctggcgg-3′ 1091–1110 501
5′-tcatacccccactggggcat-3′ 1573–1592
Lrat LRAT AF255061 5′-atctaatgcctgacatcctg-3′ 481–500 462
5′-ctagccagacatcatccaca-3′ 943–962
R1 CR1 NM_011254 5′-atgcctgtggacttcaacgg-3′ 2017–2037 408
5′-tcagtgtactttcttaaaca-3′ 2406–2425
Rdh5 11cRDH BC021372 5′-caggccaggggtcgggtggt-3′ 551–570 500
5′-tcaggagactgactgggcgg-3′ 1032–1051
Rgr RGR NM_021340 5′-atgggacacggccatccctc-3′ 411–430 504
5′-tcactgggttcggtccttct-3′ 896–915
Rl1 CRAL NM_020599 5′-acaagtatggtcgagtggtt-3′ 681–700 499
5′-tcataaggctgtgttctcaa-3′ 1161–1180
Rpe65 RPE65 NM_029987 5′-atatgtacttcctttgacaa-3′ 1100–1120 502
5′-tcaggatcttttgaacagtc-3′ 1583–1602
Stra6 STRA6 NM_009291 5′-ctcttccccatcaacatgctggt-3′ 1900–1923 450
5′-gccattggccttggcactagtaag-3′ 2326–2350
Figure 1.
 
Homologous recombination at the mouse Lrat locus produces a conditional allele and evidence that Cre-mediated recombination produces a null allele. (A, top) The mouse wild-type (WT, +) locus consists of two exons (boxes) separated by a single intron. Solid areas in exons: coding sequences; shaded areas: untranslated regions. (A, middle) In the targeted, conditional (L3) allele of the Lrat gene, the first loxP site is inserted in exon 1. The loxP-flanked PKG-NeoA+ cassette is inserted in the intron. (A, bottom) The excised (L−) allele, in which the Cre-mediated excision deletes the initiation codon ATG and the splice donor site of exon 1, as well as the entire PGK-NeoA+ cassette, making it nonfunctional (i.e., a null allele). Arrows: locations of primers P1 to P6 used for genotyping. The size of the amplified fragment is indicated for each pair of primers. (B) Genotyping of progeny from Lrat +/L3 intercrosses using primers P1 and P2. (C) Genotyping of progeny from Lrat +/L− intercrosses using primers P3 and P4. A DNA ladder (Invitrogen, San Diego, CA) is included to indicate the size of the amplified fragments (Markers). Right: the identity of the amplified fragments. (D) Western blot analysis of proteins from RPE tissues with the indicated genotypes using an anti-Lrat monoclonal antibody (top), and an anti-actin antibody (bottom). A bovine RPE extract (lane 1, RPE) is used as a positive control for Lrat immunoreactivity. Lrat is detected in WT (lane 2), but not in Lrat L−/L− (lane 3) and Lrat rpe−/− (lane 4) RPE extracts. (E) Immunohistochemical detection of Lrat on eye sections from adult female mice with the indicated genotypes. Lrat is present in the RPE cell layer of WT (left) but not of Lrat L−/L− (right) mice. The insets represent a higher magnification of an RPE cell. Note that some nonspecific staining is observed in choroidal cells of both WT and Lrat L−/L− mice. CH, choroid layer; PL photoreceptor layer; RPE, retinal pigment epithelium. Bar, 15 μm.
Figure 1.
 
Homologous recombination at the mouse Lrat locus produces a conditional allele and evidence that Cre-mediated recombination produces a null allele. (A, top) The mouse wild-type (WT, +) locus consists of two exons (boxes) separated by a single intron. Solid areas in exons: coding sequences; shaded areas: untranslated regions. (A, middle) In the targeted, conditional (L3) allele of the Lrat gene, the first loxP site is inserted in exon 1. The loxP-flanked PKG-NeoA+ cassette is inserted in the intron. (A, bottom) The excised (L−) allele, in which the Cre-mediated excision deletes the initiation codon ATG and the splice donor site of exon 1, as well as the entire PGK-NeoA+ cassette, making it nonfunctional (i.e., a null allele). Arrows: locations of primers P1 to P6 used for genotyping. The size of the amplified fragment is indicated for each pair of primers. (B) Genotyping of progeny from Lrat +/L3 intercrosses using primers P1 and P2. (C) Genotyping of progeny from Lrat +/L− intercrosses using primers P3 and P4. A DNA ladder (Invitrogen, San Diego, CA) is included to indicate the size of the amplified fragments (Markers). Right: the identity of the amplified fragments. (D) Western blot analysis of proteins from RPE tissues with the indicated genotypes using an anti-Lrat monoclonal antibody (top), and an anti-actin antibody (bottom). A bovine RPE extract (lane 1, RPE) is used as a positive control for Lrat immunoreactivity. Lrat is detected in WT (lane 2), but not in Lrat L−/L− (lane 3) and Lrat rpe−/− (lane 4) RPE extracts. (E) Immunohistochemical detection of Lrat on eye sections from adult female mice with the indicated genotypes. Lrat is present in the RPE cell layer of WT (left) but not of Lrat L−/L− (right) mice. The insets represent a higher magnification of an RPE cell. Note that some nonspecific staining is observed in choroidal cells of both WT and Lrat L−/L− mice. CH, choroid layer; PL photoreceptor layer; RPE, retinal pigment epithelium. Bar, 15 μm.
Figure 2.
 
Lrat protein detection in nonocular tissues of Lrat mutants. Western blot analysis of microsomal extracts from nonocular tissues from WT control, Lrat L−/L− and Lrat rpe−/− mice using an anti-mouse Lrat monoclonal antibody. A bovine RPE extract was included as a positive control for Lrat immunodetection. A protein band with the same molecular mass as bovine RPE is observed in all extracts from WT mice (top). The same pattern of distribution was observed in Lrat rpe−/− extracts (bottom). In contrast, no Lrat was detected in extracts from Lrat L−/L− samples (middle).
Figure 2.
 
Lrat protein detection in nonocular tissues of Lrat mutants. Western blot analysis of microsomal extracts from nonocular tissues from WT control, Lrat L−/L− and Lrat rpe−/− mice using an anti-mouse Lrat monoclonal antibody. A bovine RPE extract was included as a positive control for Lrat immunodetection. A protein band with the same molecular mass as bovine RPE is observed in all extracts from WT mice (top). The same pattern of distribution was observed in Lrat rpe−/− extracts (bottom). In contrast, no Lrat was detected in extracts from Lrat L−/L− samples (middle).
Figure 3.
 
PCR analysis of Lrat Cre-mediated excision. PCR amplification was performed on genomic DNA extracted from RPE, brain, heart, liver, lung, kidney, testis, and pancreas of Lrat L3/L3, Lrat rpe−/−, and Lrat L−/L− mice. A set of primers P1-P2 for detection of L3 allele (423 bp) and P3-P4 for detection of the L− allele (346 bp) were used during the amplification. Because of Cre excision of the loxP-flanked region exclusively in the RPE of Lrat rpe−/− mice, the P3-P4 PCR product is the same that from Lrat L−/L− mice in which excision has taken place in all tissues.
Figure 3.
 
PCR analysis of Lrat Cre-mediated excision. PCR amplification was performed on genomic DNA extracted from RPE, brain, heart, liver, lung, kidney, testis, and pancreas of Lrat L3/L3, Lrat rpe−/−, and Lrat L−/L− mice. A set of primers P1-P2 for detection of L3 allele (423 bp) and P3-P4 for detection of the L− allele (346 bp) were used during the amplification. Because of Cre excision of the loxP-flanked region exclusively in the RPE of Lrat rpe−/− mice, the P3-P4 PCR product is the same that from Lrat L−/L− mice in which excision has taken place in all tissues.
Figure 4.
 
Histologic features of the retina in Lrat mutants. One-micrometer sections of retina from mice with the indicated genotypes are shown. Shortening of the rod outer segments and a slight reduction in the number of photoreceptor nuclei is observed in the retina of mice lacking Lrat (i.e., Lrat L−/L− and Lrat rpe−/− mutants). IS, inner segment; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Bar, 20 μm.
Figure 4.
 
Histologic features of the retina in Lrat mutants. One-micrometer sections of retina from mice with the indicated genotypes are shown. Shortening of the rod outer segments and a slight reduction in the number of photoreceptor nuclei is observed in the retina of mice lacking Lrat (i.e., Lrat L−/L− and Lrat rpe−/− mutants). IS, inner segment; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Bar, 20 μm.
Figure 5.
 
ERG analysis of Lrat mutants. ERG responses to blue-flash stimuli recorded in dark-adapted conditions are shown for each group of mice. (A, B) Average response to intensities ranging from −4.7 to −0.44 log cd-s/m2, as indicated for the WT mouse (A). (C, D) Records for only the brightest stimuli. ERG responses were similar for the WT (A) and Lrat L3/L3 (B) mice with parameters that are within normal limits. ERG responses were nondetectable for the representative Lrat L−/L− mouse (C). However, a barely detectable response was recordable in the Lrat rpe−/− mutants at the brightest flash intensities (D, asterisk). To confirm the existence of this signal, ERGs were recorded in response to 10-Hz flashed stimuli (blue, −0.87 log cd-s/m2) and analyzed by FFT, to derive the amplitude of signal and noise at the stimulus frequency. Representative waveforms are shown in (E) for WT, Lrat L−/L−, and Lrat rpe−/− mice. The FFT amplitude for the Lrat rpe−/− mouse exceeded that of noise but did not for the Lrat L−/L−, thereby confirming the existence of a very small residual response in the Lrat rpe−/− line.
Figure 5.
 
ERG analysis of Lrat mutants. ERG responses to blue-flash stimuli recorded in dark-adapted conditions are shown for each group of mice. (A, B) Average response to intensities ranging from −4.7 to −0.44 log cd-s/m2, as indicated for the WT mouse (A). (C, D) Records for only the brightest stimuli. ERG responses were similar for the WT (A) and Lrat L3/L3 (B) mice with parameters that are within normal limits. ERG responses were nondetectable for the representative Lrat L−/L− mouse (C). However, a barely detectable response was recordable in the Lrat rpe−/− mutants at the brightest flash intensities (D, asterisk). To confirm the existence of this signal, ERGs were recorded in response to 10-Hz flashed stimuli (blue, −0.87 log cd-s/m2) and analyzed by FFT, to derive the amplitude of signal and noise at the stimulus frequency. Representative waveforms are shown in (E) for WT, Lrat L−/L−, and Lrat rpe−/− mice. The FFT amplitude for the Lrat rpe−/− mouse exceeded that of noise but did not for the Lrat L−/L−, thereby confirming the existence of a very small residual response in the Lrat rpe−/− line.
Table 4.
 
Retinoid Contents in Tissues from Lrat Mutants
Table 4.
 
Retinoid Contents in Tissues from Lrat Mutants
Tissue Retinoid (nM/g) Lrat Genotype
WT (+/+) L/L L3/L3 RPE−/−
RPE all-trans RE 0.21 0.02 0.48 0.09
all-trans ROL 0.10 0.07 0.14 0.05
Liver all-trans RE 5266 1.64 3257 1642
all-trans ROL 85.96 1.01 39.76 14.71
Lung all-trans RE 1000 0.93 736 577
all-trans ROL 8.20 0.53 3.40 5.68
Testis all-trans RE 0.88 ND 0.57 0.55
all-trans ROL 0.24 0.54 0.27 0.46
Kidney all-trans RE 0.48 0.17 0.28 0.31
all-trans ROL 0.69 0.57 0.86 1.76
Figure 6.
 
HPLC analysis of retinoid extracts from Lrat mutants. Chromatographic separation by HPLC of nonpolar retinoids from RPE extracts of Lrat L−/L− and Lrat rpe−/− mice compared with that of WT and Lrat L3/L3 controls, respectively. Peaks corresponding to all-trans retinyl esters and all-trans retinol are indicated. All-trans retinyl esters were not observed in Lrat L−/L− RPE tissues, whereas a minor ester peak was detected in RPE tissues from Lrat rpe−/− mice.
Figure 6.
 
HPLC analysis of retinoid extracts from Lrat mutants. Chromatographic separation by HPLC of nonpolar retinoids from RPE extracts of Lrat L−/L− and Lrat rpe−/− mice compared with that of WT and Lrat L3/L3 controls, respectively. Peaks corresponding to all-trans retinyl esters and all-trans retinol are indicated. All-trans retinyl esters were not observed in Lrat L−/L− RPE tissues, whereas a minor ester peak was detected in RPE tissues from Lrat rpe−/− mice.
Figure 7.
 
Analysis of RNA transcripts encoding genes involved in the visual cycle. RT-PCR amplification of Rpe65, Lrat, Rdh5, Rlbp1, Rgr, Rbp1, Dgat1, and Stra6 RNA transcripts in WT, Lrat L−/L−, and Lrat rpe−/− RPE tissues. Amplification was performed using the set of primers described in Table 3 .
Figure 7.
 
Analysis of RNA transcripts encoding genes involved in the visual cycle. RT-PCR amplification of Rpe65, Lrat, Rdh5, Rlbp1, Rgr, Rbp1, Dgat1, and Stra6 RNA transcripts in WT, Lrat L−/L−, and Lrat rpe−/− RPE tissues. Amplification was performed using the set of primers described in Table 3 .
Figure 8.
 
Western blot analysis of Stra6 and Dgat1 in RPE tissues from control mice and Lrat mutants. Top: expression of Stra6 protein in RPE samples from WT (Lrat +/+), Lrat L−/L−, Lrat L3/L3, and Lrat rpe−/− mice. A single band of ∼74 kDa was observed in all samples analyzed. No difference in the level of expression was observed in Lrat mutants and control samples (lanes 14). Similar results were found in protein extracts from testis of the same set of animals and are included for comparison (lanes 58). Bottom: single band of ∼57 kDa was obtained in all samples analyzed. This analysis showed similar levels of expression of Dgat1 protein in RPE samples from control mice and Lrat mutants (lanes 14). Also, no difference was found in the levels of Dgat1 in WAT samples from Lrat mutants and control mice (lanes 58). However, despite loading equal amounts of protein in each track, WAT samples had stronger Dgat1 signal compared with RPE samples.
Figure 8.
 
Western blot analysis of Stra6 and Dgat1 in RPE tissues from control mice and Lrat mutants. Top: expression of Stra6 protein in RPE samples from WT (Lrat +/+), Lrat L−/L−, Lrat L3/L3, and Lrat rpe−/− mice. A single band of ∼74 kDa was observed in all samples analyzed. No difference in the level of expression was observed in Lrat mutants and control samples (lanes 14). Similar results were found in protein extracts from testis of the same set of animals and are included for comparison (lanes 58). Bottom: single band of ∼57 kDa was obtained in all samples analyzed. This analysis showed similar levels of expression of Dgat1 protein in RPE samples from control mice and Lrat mutants (lanes 14). Also, no difference was found in the levels of Dgat1 in WAT samples from Lrat mutants and control mice (lanes 58). However, despite loading equal amounts of protein in each track, WAT samples had stronger Dgat1 signal compared with RPE samples.
Figure 9.
 
Western blot analysis of proteins involved in the visual cycle in RPE tissue from control mice and Lrat rpe−/− mutants. (A) Comparison of the expression levels of Lrat, Crbp1, Rpe65, Rgr, and Cralbp in RPE between control (Lrat L3/L3, left lanes) and mutant (Lrat rpe−/−, right lanes) mice. The sizes of the immunodetected proteins are indicated in parentheses. (B) After they were stripped, the membranes were reacted with an anti-actin monoclonal antibody to confirm an equal amount of protein loaded in both groups of mice.
Figure 9.
 
Western blot analysis of proteins involved in the visual cycle in RPE tissue from control mice and Lrat rpe−/− mutants. (A) Comparison of the expression levels of Lrat, Crbp1, Rpe65, Rgr, and Cralbp in RPE between control (Lrat L3/L3, left lanes) and mutant (Lrat rpe−/−, right lanes) mice. The sizes of the immunodetected proteins are indicated in parentheses. (B) After they were stripped, the membranes were reacted with an anti-actin monoclonal antibody to confirm an equal amount of protein loaded in both groups of mice.
The authors thank Krzysztof Palczewski and Alexander R. Moise (Case Western University, Cleveland, OH) for providing the monoclonal anti-Lrat antibody; T. Michael Redmond (Laboratory of Retinal Cell and Molecular Biology National Eye Institute, Bethesda, MD) for the anti-RPE65 antibody; David Ong (Vanderbilt University School of Medicine, Nashville, TN) for the anti-CRBP1 antibody; Henry Fong (University of Southern California, Los Angeles, CA) for the anti-RGR antibody; Robert V. Farese, Jr (University of California, San Francisco, CA) for the anti-DGAT1 antibody; Pamela Stiles, Michael Chan, Ginda Phongadith (Jules Stein Eye Institute, UCLA) and Betty Feret (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université Louis Pasteur, Strasbourg, France) for excellent technical assistance. 
BokD. The retinal pigment epithelium: a versatile partner in vision. J Cell Sci. 1993;17(suppl.)189–195.
McBeeJK, PalczewskiK, BaehrW, PepperbergDR. Confronting complexity: the interlink of phototransduction and retinoid metabolism in the vertebrate retina. Prog Retin Eye Res. 2001;20:469–529. [CrossRef] [PubMed]
LambTD, PughEN, Jr. Dark adaptation and the retinol cycle of vision. Prog Retin Eye Res. 2004;23:307–380. [CrossRef] [PubMed]
GoodmanDS. Plasma retino-binding protein.SpornMB RobertsAB GoodmanDS eds. The Retinoids. 1984;2:42–88.Academic Press New York.
HellerM, BokD. A specific receptor for retinal binding protein as detected by the binding of human and bovine binding protein to pigment epithelial cells. Am J Ophthalmol. 1976;81:93–97. [CrossRef] [PubMed]
KawaguchiR, YuJ, HondaJ, et al. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science. 2007;315:820–825. [CrossRef] [PubMed]
HerrFM, OngDE. Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins. Biochemistry. 1992;31:6748–6755. [CrossRef] [PubMed]
MacDonaldPN, OngDE. Evidence for a lecithin-retinol acyltransferase activity in the rat small intestine. J Biol Chem. 1988;263:12478–12482. [PubMed]
SaariJC, BredbergDL. Lecithin:retinal acyltransferase in retinal pigment epithelial microsomes. J Biol Chem. 1989;264:8636–8640. [PubMed]
MoiseyevG, ChenY, TakahashiY, WuBX, MaJX. RPE65 is the isomerohydrolase in the retinoid visual cycle. Proc Natl Acad Sci USA. 2005;102:12413–12418. [CrossRef] [PubMed]
JinM, LiS, MoghrabiWN, SunH, TravisGH. Rpe65 is the retinal isomerase in bovine retinal pigment epithelium. Cell. 2005;122:449–459. [CrossRef] [PubMed]
RedmondTM, PoliakovE, YuS, TsaiJY, LuZ, GentlemanS. Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci USA. 2005;102:13658–13663. [CrossRef] [PubMed]
SaariJC, BredbergDL. Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina. J Biol Chem. 1987;262:7618–7622. [PubMed]
CarlsonA, BokD. Promotion of the release of 11-cis-retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid binding protein. Biochem. 1992;31:9056–9062. [CrossRef]
OngDE, MacDonaldPN, GubitosiAM. Esterification of retinol in rat liver. J Biol Chem. 1988;263:5789–5796. [PubMed]
SchmittYQ, OngDE. Expression of cellular retinol-binding protein and lecithin-retinol acyltransferase in developing rat testis. Biol Reprod. 1993;49:972–979. [CrossRef] [PubMed]
MawMA, KennedyB, KnightA, et al. Mutation of the gene encoding cellular retinaldehyde-binding protein in autosomal recessive retinitis pigmentosa. Nat Genet. 1997;17:198–200. [CrossRef] [PubMed]
GuSM, ThompsonDA, SrikumariCR, et al. Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. Nat Genet. 1997;17:194–197. [CrossRef] [PubMed]
MarlhensF, BareilC, GriffoinJM, et al. Mutations in RPE65 cause Leber’s congenital amaurosis. Nat Genet. 1997;17:139–141. [CrossRef] [PubMed]
YamamotoH, SimonA, ErikssonU, HarrisE, BersonEL, DryjaTP. Mutations in the gene encoding 11-cis retinal dehydrogenase cause delayed dark adaptation and fundus albipunctatus. Nat Genet. 1999;22:188–191. [CrossRef] [PubMed]
NakamuraM, HottaY, TanikawaA, TerasakiH, MiyakeY. A high association with cone dystrophy in fundus albipunctatus caused by mutations of the RDH5 gene. Invest Ophthalmol Vis Sci. 2000;41:3925–3932. [PubMed]
MorimuraH, Saindelle-RibeaudeauF, BersonEL, DryjaTP. Mutations in RGR, encoding a light-sensitive opsin homologue, in patients with retinitis pigmentosa. Nat Genet. 1999;23:393–394. [CrossRef] [PubMed]
ThompsonDA, LiY, McHenryCL, et al. Mutations in the gene encoding Lecithin retinal acyltransferase are associated with early-onset severe retinal dystrophy. Nat Genet. 2001;28:123–124. [CrossRef] [PubMed]
BattenML, ImanishiY, MaedaT, et al. Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. J Biol Chem. 2004;279:10422–10432. [CrossRef] [PubMed]
LiuL, GudasLJ. Disruption of the lecithin:retinal acyltransferase gene makes mice more susceptible to vitamin A deficiency. J Biol Chem. 2005;280:40226–40234. [CrossRef] [PubMed]
NapoliJL. Biochemical pathways of retinoid transport, metabolism, and signal transduction. Clin Immunol Immunopathol. 1996;80:S52–S56. [CrossRef] [PubMed]
NagyA. Cre recombinase: the universal reagent for genome tailoring. Genesis. 2000;26:99–109. [CrossRef] [PubMed]
MetzgerD, ChambonP. Site- and time-specific gene targeting in the mouse. Methods. 2001;24:71–80. [CrossRef] [PubMed]
DierichA, DolleP. Gene targeting in embryonic stem cells.KlugS ThielR eds. Methods in Development Biology/Toxicology. 1997;111–123.Blackwell Sciences Oxford, UK.
IndraAK, WarotX, BrocardJ, et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 1999;27:4324–4327. [CrossRef] [PubMed]
PittlerS, BaehrW. Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse. Proc Natl Acad Sci USA. 1991;88:8322–8326. [CrossRef] [PubMed]
TaketoM, SchroederA, MobraatenL, et al. FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc Natl Acad Sci USA. 1991;88:2065–2069. [CrossRef] [PubMed]
MataN, MoghrabiWN, LeeJS, et al. Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells. J Biol Chem. 2003;279:635–643. [PubMed]
TaoL, ShenD, PandeyS, HaoW, RichKA, FongHKW. Structure and developmental expression of the mouse RGR opsin gene. Mol Vis. 1998;4:25. [PubMed]
BokD, OngDE, ChytilF. Immunocytochemical localization of cellular retinal binding protein in the rat retina. Invest Ophthalmol Vis Sci. 1984;25:877–883. [PubMed]
LinJJ-C. Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells. Proc Natl Acad Sci USA. 1991;78:2335–2339.
RedmondTM, HamelCP. Genetic analysis of RPE65: from human disease to mouse model. Methods Enzymol. 2000;316:705–724. [PubMed]
BouilletP, SapinV, ChazaudC, et al. Developmental expression pattern of stra6, a retinoic aci-responsive gene encoding a new type of membrane protein. Mech Dev. 1997;63:173–186. [CrossRef] [PubMed]
ChenHC, StoneSJ, ZhouP, BuhmanKK, FareseRV, Jr. Dissociation of obesity and impaired glucose disposal in mice overexpressing acyl coenzyme. A: diacylglycerol acyltransferase 1 in white adipose tissue. Diabetes. 2002;51:3189–3195. [CrossRef] [PubMed]
VernetN, DennefeldC, Rochette-EglyC, et al. Retinoic acid metabolism and signaling pathways in the adult and developing mouse testis. Endocrinology. 2006;147:96–110. [CrossRef] [PubMed]
BeermanF. The tyrosinase related protein-1 (Tyrp1) promoter in transgenic experiments: targeted expression to the retinal pigment epithelium. Cell Mol Biol. 1999;45:961–968. [PubMed]
MoriM, MetzgerD, GarnierJM, ChambonP, MarkM. Site-specific somatic mutagenesis in the retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2002;43:1384–1388. [PubMed]
O’ByrneSM, WongsirirojN, LibienJ, et al. Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT). J Biol Chem. 2005;280:35647–35657. [CrossRef] [PubMed]
CasesS, SmithSJ, ZhengYW, et al. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransfersase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci USA. 1998;95:13018–13023. [CrossRef] [PubMed]
WenzelA, OberhauserV, PughEN, et al. The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light. J Biol Chem. 2005;280:29874–29884. [CrossRef] [PubMed]
RuizA, WinstonA, LimYH, GilbertBA, RandoRR, BokD. Molecular and biochemical characterization of lecithin retinol acyltransferase. J Biol Chem. 1999;274:3834–3841. [CrossRef] [PubMed]
ZolfaghariR, RossAC. Lecithin:retinol acyltransferase from mouse and rat liver. cDNA cloning and liver-specific regulation by dietary vitamin A and retinoic acid. J Lipid Res. 2000;41:2024–2034. [PubMed]
RandoRR. The biochemistry of the visual cycle. Chem Rev. 2001;101:1881–1896. [CrossRef] [PubMed]
WinstonA, RandoRR. Regulation of isomerohydrolase activity in the visual cycle. Biochemistry. 1998;37:2044–2050. [CrossRef] [PubMed]
MoriM, MetzgerD, PicaudS, et al. Retinal dystrophy resulting from ablation of RXRa in the mouse retinal pigment epithelium. Am J Pathol. 2004;164:701–710. [CrossRef] [PubMed]
SaariJC, BredbergDL. CoA- and non CoA-dependent retinol esterification in retinal pigment epithelium. J Biol Chem. 1988;263:8084–8090. [PubMed]
KaschulaCH, JinMH, Desmond-SmithNS, TravisGH. Acyl CoA:retinol acyltransferase (ARAT) activity is present in bovine retinal pigment epithelium. Exp Eye Res. 2006;82:111–121. [CrossRef] [PubMed]
RandolphRK, WinkerKE, RossAC. Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes. Arch Biochem Biophys. 1991;288:500–508. [CrossRef] [PubMed]
YenCL, MonettiM, BurriBJ, FareseRV, Jr. The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerol, waxes, and retinyl esters. J Lipid Res. 2005;46:1502–1511. [CrossRef] [PubMed]
ChenHC, FareseRV, Jr. Inhibition of triglyceride synthesis as a treatment strategy for obesity: lessons from DGAT1-deficient mice. Arterioscler Thromb Vasc Biol. 2005;25:482–486. [CrossRef] [PubMed]
GoodmanDS, BlanerWS. Biosynthesis, absorption, and hepatic metabolism of retinol.SpornMB RobertsAB GoodmanDS eds. The Retinoids. 1984;2:2–34.Academic Press New York.
XueL, JahngWJ, GollapalliD, RandoRR. Palmitoyl transferase activity of lecithin retinol acyltransferase. Biochemistry. 2006;45:10710–10718. [CrossRef] [PubMed]
Figure 1.
 
Homologous recombination at the mouse Lrat locus produces a conditional allele and evidence that Cre-mediated recombination produces a null allele. (A, top) The mouse wild-type (WT, +) locus consists of two exons (boxes) separated by a single intron. Solid areas in exons: coding sequences; shaded areas: untranslated regions. (A, middle) In the targeted, conditional (L3) allele of the Lrat gene, the first loxP site is inserted in exon 1. The loxP-flanked PKG-NeoA+ cassette is inserted in the intron. (A, bottom) The excised (L−) allele, in which the Cre-mediated excision deletes the initiation codon ATG and the splice donor site of exon 1, as well as the entire PGK-NeoA+ cassette, making it nonfunctional (i.e., a null allele). Arrows: locations of primers P1 to P6 used for genotyping. The size of the amplified fragment is indicated for each pair of primers. (B) Genotyping of progeny from Lrat +/L3 intercrosses using primers P1 and P2. (C) Genotyping of progeny from Lrat +/L− intercrosses using primers P3 and P4. A DNA ladder (Invitrogen, San Diego, CA) is included to indicate the size of the amplified fragments (Markers). Right: the identity of the amplified fragments. (D) Western blot analysis of proteins from RPE tissues with the indicated genotypes using an anti-Lrat monoclonal antibody (top), and an anti-actin antibody (bottom). A bovine RPE extract (lane 1, RPE) is used as a positive control for Lrat immunoreactivity. Lrat is detected in WT (lane 2), but not in Lrat L−/L− (lane 3) and Lrat rpe−/− (lane 4) RPE extracts. (E) Immunohistochemical detection of Lrat on eye sections from adult female mice with the indicated genotypes. Lrat is present in the RPE cell layer of WT (left) but not of Lrat L−/L− (right) mice. The insets represent a higher magnification of an RPE cell. Note that some nonspecific staining is observed in choroidal cells of both WT and Lrat L−/L− mice. CH, choroid layer; PL photoreceptor layer; RPE, retinal pigment epithelium. Bar, 15 μm.
Figure 1.
 
Homologous recombination at the mouse Lrat locus produces a conditional allele and evidence that Cre-mediated recombination produces a null allele. (A, top) The mouse wild-type (WT, +) locus consists of two exons (boxes) separated by a single intron. Solid areas in exons: coding sequences; shaded areas: untranslated regions. (A, middle) In the targeted, conditional (L3) allele of the Lrat gene, the first loxP site is inserted in exon 1. The loxP-flanked PKG-NeoA+ cassette is inserted in the intron. (A, bottom) The excised (L−) allele, in which the Cre-mediated excision deletes the initiation codon ATG and the splice donor site of exon 1, as well as the entire PGK-NeoA+ cassette, making it nonfunctional (i.e., a null allele). Arrows: locations of primers P1 to P6 used for genotyping. The size of the amplified fragment is indicated for each pair of primers. (B) Genotyping of progeny from Lrat +/L3 intercrosses using primers P1 and P2. (C) Genotyping of progeny from Lrat +/L− intercrosses using primers P3 and P4. A DNA ladder (Invitrogen, San Diego, CA) is included to indicate the size of the amplified fragments (Markers). Right: the identity of the amplified fragments. (D) Western blot analysis of proteins from RPE tissues with the indicated genotypes using an anti-Lrat monoclonal antibody (top), and an anti-actin antibody (bottom). A bovine RPE extract (lane 1, RPE) is used as a positive control for Lrat immunoreactivity. Lrat is detected in WT (lane 2), but not in Lrat L−/L− (lane 3) and Lrat rpe−/− (lane 4) RPE extracts. (E) Immunohistochemical detection of Lrat on eye sections from adult female mice with the indicated genotypes. Lrat is present in the RPE cell layer of WT (left) but not of Lrat L−/L− (right) mice. The insets represent a higher magnification of an RPE cell. Note that some nonspecific staining is observed in choroidal cells of both WT and Lrat L−/L− mice. CH, choroid layer; PL photoreceptor layer; RPE, retinal pigment epithelium. Bar, 15 μm.
Figure 2.
 
Lrat protein detection in nonocular tissues of Lrat mutants. Western blot analysis of microsomal extracts from nonocular tissues from WT control, Lrat L−/L− and Lrat rpe−/− mice using an anti-mouse Lrat monoclonal antibody. A bovine RPE extract was included as a positive control for Lrat immunodetection. A protein band with the same molecular mass as bovine RPE is observed in all extracts from WT mice (top). The same pattern of distribution was observed in Lrat rpe−/− extracts (bottom). In contrast, no Lrat was detected in extracts from Lrat L−/L− samples (middle).
Figure 2.
 
Lrat protein detection in nonocular tissues of Lrat mutants. Western blot analysis of microsomal extracts from nonocular tissues from WT control, Lrat L−/L− and Lrat rpe−/− mice using an anti-mouse Lrat monoclonal antibody. A bovine RPE extract was included as a positive control for Lrat immunodetection. A protein band with the same molecular mass as bovine RPE is observed in all extracts from WT mice (top). The same pattern of distribution was observed in Lrat rpe−/− extracts (bottom). In contrast, no Lrat was detected in extracts from Lrat L−/L− samples (middle).
Figure 3.
 
PCR analysis of Lrat Cre-mediated excision. PCR amplification was performed on genomic DNA extracted from RPE, brain, heart, liver, lung, kidney, testis, and pancreas of Lrat L3/L3, Lrat rpe−/−, and Lrat L−/L− mice. A set of primers P1-P2 for detection of L3 allele (423 bp) and P3-P4 for detection of the L− allele (346 bp) were used during the amplification. Because of Cre excision of the loxP-flanked region exclusively in the RPE of Lrat rpe−/− mice, the P3-P4 PCR product is the same that from Lrat L−/L− mice in which excision has taken place in all tissues.
Figure 3.
 
PCR analysis of Lrat Cre-mediated excision. PCR amplification was performed on genomic DNA extracted from RPE, brain, heart, liver, lung, kidney, testis, and pancreas of Lrat L3/L3, Lrat rpe−/−, and Lrat L−/L− mice. A set of primers P1-P2 for detection of L3 allele (423 bp) and P3-P4 for detection of the L− allele (346 bp) were used during the amplification. Because of Cre excision of the loxP-flanked region exclusively in the RPE of Lrat rpe−/− mice, the P3-P4 PCR product is the same that from Lrat L−/L− mice in which excision has taken place in all tissues.
Figure 4.
 
Histologic features of the retina in Lrat mutants. One-micrometer sections of retina from mice with the indicated genotypes are shown. Shortening of the rod outer segments and a slight reduction in the number of photoreceptor nuclei is observed in the retina of mice lacking Lrat (i.e., Lrat L−/L− and Lrat rpe−/− mutants). IS, inner segment; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Bar, 20 μm.
Figure 4.
 
Histologic features of the retina in Lrat mutants. One-micrometer sections of retina from mice with the indicated genotypes are shown. Shortening of the rod outer segments and a slight reduction in the number of photoreceptor nuclei is observed in the retina of mice lacking Lrat (i.e., Lrat L−/L− and Lrat rpe−/− mutants). IS, inner segment; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium. Bar, 20 μm.
Figure 5.
 
ERG analysis of Lrat mutants. ERG responses to blue-flash stimuli recorded in dark-adapted conditions are shown for each group of mice. (A, B) Average response to intensities ranging from −4.7 to −0.44 log cd-s/m2, as indicated for the WT mouse (A). (C, D) Records for only the brightest stimuli. ERG responses were similar for the WT (A) and Lrat L3/L3 (B) mice with parameters that are within normal limits. ERG responses were nondetectable for the representative Lrat L−/L− mouse (C). However, a barely detectable response was recordable in the Lrat rpe−/− mutants at the brightest flash intensities (D, asterisk). To confirm the existence of this signal, ERGs were recorded in response to 10-Hz flashed stimuli (blue, −0.87 log cd-s/m2) and analyzed by FFT, to derive the amplitude of signal and noise at the stimulus frequency. Representative waveforms are shown in (E) for WT, Lrat L−/L−, and Lrat rpe−/− mice. The FFT amplitude for the Lrat rpe−/− mouse exceeded that of noise but did not for the Lrat L−/L−, thereby confirming the existence of a very small residual response in the Lrat rpe−/− line.
Figure 5.
 
ERG analysis of Lrat mutants. ERG responses to blue-flash stimuli recorded in dark-adapted conditions are shown for each group of mice. (A, B) Average response to intensities ranging from −4.7 to −0.44 log cd-s/m2, as indicated for the WT mouse (A). (C, D) Records for only the brightest stimuli. ERG responses were similar for the WT (A) and Lrat L3/L3 (B) mice with parameters that are within normal limits. ERG responses were nondetectable for the representative Lrat L−/L− mouse (C). However, a barely detectable response was recordable in the Lrat rpe−/− mutants at the brightest flash intensities (D, asterisk). To confirm the existence of this signal, ERGs were recorded in response to 10-Hz flashed stimuli (blue, −0.87 log cd-s/m2) and analyzed by FFT, to derive the amplitude of signal and noise at the stimulus frequency. Representative waveforms are shown in (E) for WT, Lrat L−/L−, and Lrat rpe−/− mice. The FFT amplitude for the Lrat rpe−/− mouse exceeded that of noise but did not for the Lrat L−/L−, thereby confirming the existence of a very small residual response in the Lrat rpe−/− line.
Figure 6.
 
HPLC analysis of retinoid extracts from Lrat mutants. Chromatographic separation by HPLC of nonpolar retinoids from RPE extracts of Lrat L−/L− and Lrat rpe−/− mice compared with that of WT and Lrat L3/L3 controls, respectively. Peaks corresponding to all-trans retinyl esters and all-trans retinol are indicated. All-trans retinyl esters were not observed in Lrat L−/L− RPE tissues, whereas a minor ester peak was detected in RPE tissues from Lrat rpe−/− mice.
Figure 6.
 
HPLC analysis of retinoid extracts from Lrat mutants. Chromatographic separation by HPLC of nonpolar retinoids from RPE extracts of Lrat L−/L− and Lrat rpe−/− mice compared with that of WT and Lrat L3/L3 controls, respectively. Peaks corresponding to all-trans retinyl esters and all-trans retinol are indicated. All-trans retinyl esters were not observed in Lrat L−/L− RPE tissues, whereas a minor ester peak was detected in RPE tissues from Lrat rpe−/− mice.
Figure 7.
 
Analysis of RNA transcripts encoding genes involved in the visual cycle. RT-PCR amplification of Rpe65, Lrat, Rdh5, Rlbp1, Rgr, Rbp1, Dgat1, and Stra6 RNA transcripts in WT, Lrat L−/L−, and Lrat rpe−/− RPE tissues. Amplification was performed using the set of primers described in Table 3 .
Figure 7.
 
Analysis of RNA transcripts encoding genes involved in the visual cycle. RT-PCR amplification of Rpe65, Lrat, Rdh5, Rlbp1, Rgr, Rbp1, Dgat1, and Stra6 RNA transcripts in WT, Lrat L−/L−, and Lrat rpe−/− RPE tissues. Amplification was performed using the set of primers described in Table 3 .
Figure 8.
 
Western blot analysis of Stra6 and Dgat1 in RPE tissues from control mice and Lrat mutants. Top: expression of Stra6 protein in RPE samples from WT (Lrat +/+), Lrat L−/L−, Lrat L3/L3, and Lrat rpe−/− mice. A single band of ∼74 kDa was observed in all samples analyzed. No difference in the level of expression was observed in Lrat mutants and control samples (lanes 14). Similar results were found in protein extracts from testis of the same set of animals and are included for comparison (lanes 58). Bottom: single band of ∼57 kDa was obtained in all samples analyzed. This analysis showed similar levels of expression of Dgat1 protein in RPE samples from control mice and Lrat mutants (lanes 14). Also, no difference was found in the levels of Dgat1 in WAT samples from Lrat mutants and control mice (lanes 58). However, despite loading equal amounts of protein in each track, WAT samples had stronger Dgat1 signal compared with RPE samples.
Figure 8.
 
Western blot analysis of Stra6 and Dgat1 in RPE tissues from control mice and Lrat mutants. Top: expression of Stra6 protein in RPE samples from WT (Lrat +/+), Lrat L−/L−, Lrat L3/L3, and Lrat rpe−/− mice. A single band of ∼74 kDa was observed in all samples analyzed. No difference in the level of expression was observed in Lrat mutants and control samples (lanes 14). Similar results were found in protein extracts from testis of the same set of animals and are included for comparison (lanes 58). Bottom: single band of ∼57 kDa was obtained in all samples analyzed. This analysis showed similar levels of expression of Dgat1 protein in RPE samples from control mice and Lrat mutants (lanes 14). Also, no difference was found in the levels of Dgat1 in WAT samples from Lrat mutants and control mice (lanes 58). However, despite loading equal amounts of protein in each track, WAT samples had stronger Dgat1 signal compared with RPE samples.
Figure 9.
 
Western blot analysis of proteins involved in the visual cycle in RPE tissue from control mice and Lrat rpe−/− mutants. (A) Comparison of the expression levels of Lrat, Crbp1, Rpe65, Rgr, and Cralbp in RPE between control (Lrat L3/L3, left lanes) and mutant (Lrat rpe−/−, right lanes) mice. The sizes of the immunodetected proteins are indicated in parentheses. (B) After they were stripped, the membranes were reacted with an anti-actin monoclonal antibody to confirm an equal amount of protein loaded in both groups of mice.
Figure 9.
 
Western blot analysis of proteins involved in the visual cycle in RPE tissue from control mice and Lrat rpe−/− mutants. (A) Comparison of the expression levels of Lrat, Crbp1, Rpe65, Rgr, and Cralbp in RPE between control (Lrat L3/L3, left lanes) and mutant (Lrat rpe−/−, right lanes) mice. The sizes of the immunodetected proteins are indicated in parentheses. (B) After they were stripped, the membranes were reacted with an anti-actin monoclonal antibody to confirm an equal amount of protein loaded in both groups of mice.
Table 1.
 
Sequence of Primers Used for Genotype Analysis
Table 1.
 
Sequence of Primers Used for Genotype Analysis
Name Sequence Gene Expected Size of Allele (bp)
WT(+) L3 L−
P1 5′-aggagaagtcagggtgcaga-3′ Lrat 376 423 NA
P2 5′-tcatcctaaggcaaccaacc-3′
P3 5′-gcactttggcttctcctctg-3′ Lrat 877 346
P4 5′-gtggccacacccttttattg-3′
P5 5′-ggttctccggccgcttgggt-3′ PGK-NeoA+ NA* 741 NA
P6 5′-gaaggcgatgcgctgcgaat-3′
Table 2.
 
Antibodies Used for Western Blot Analysis
Table 2.
 
Antibodies Used for Western Blot Analysis
Protein Calculated MW (kDa) Antibodies Antigen Origin Source Reference
ACTIN 43 Monoclonal Rat Lin 36
CRALBP 36 Polyclonal Bovine Bok Unpublished (1984)
CRBP1 16 Polyclonal Rat Bok et al. 35
DGAT1 57 Polyclonal Mouse Chen et al. 39
LRAT 25 Monoclonal Mouse Batten et al. 24
RGR 32 Polyclonal Mouse Tao et al. 34
RPE65 61 Polyclonal Bovine Redmond & Hamel 37
STRA6 74 Polyclonal Mouse Bouillet et al. 38
Table 3.
 
Sequence of Primers Used for RT-PCR Analysis
Table 3.
 
Sequence of Primers Used for RT-PCR Analysis
Gene Protein Accession Number Sequence Position Size (bp)
Dgat1 DGAT1 AF078752 5′-gcgtctcttaaagctggcgg-3′ 1091–1110 501
5′-tcatacccccactggggcat-3′ 1573–1592
Lrat LRAT AF255061 5′-atctaatgcctgacatcctg-3′ 481–500 462
5′-ctagccagacatcatccaca-3′ 943–962
R1 CR1 NM_011254 5′-atgcctgtggacttcaacgg-3′ 2017–2037 408
5′-tcagtgtactttcttaaaca-3′ 2406–2425
Rdh5 11cRDH BC021372 5′-caggccaggggtcgggtggt-3′ 551–570 500
5′-tcaggagactgactgggcgg-3′ 1032–1051
Rgr RGR NM_021340 5′-atgggacacggccatccctc-3′ 411–430 504
5′-tcactgggttcggtccttct-3′ 896–915
Rl1 CRAL NM_020599 5′-acaagtatggtcgagtggtt-3′ 681–700 499
5′-tcataaggctgtgttctcaa-3′ 1161–1180
Rpe65 RPE65 NM_029987 5′-atatgtacttcctttgacaa-3′ 1100–1120 502
5′-tcaggatcttttgaacagtc-3′ 1583–1602
Stra6 STRA6 NM_009291 5′-ctcttccccatcaacatgctggt-3′ 1900–1923 450
5′-gccattggccttggcactagtaag-3′ 2326–2350
Table 4.
 
Retinoid Contents in Tissues from Lrat Mutants
Table 4.
 
Retinoid Contents in Tissues from Lrat Mutants
Tissue Retinoid (nM/g) Lrat Genotype
WT (+/+) L/L L3/L3 RPE−/−
RPE all-trans RE 0.21 0.02 0.48 0.09
all-trans ROL 0.10 0.07 0.14 0.05
Liver all-trans RE 5266 1.64 3257 1642
all-trans ROL 85.96 1.01 39.76 14.71
Lung all-trans RE 1000 0.93 736 577
all-trans ROL 8.20 0.53 3.40 5.68
Testis all-trans RE 0.88 ND 0.57 0.55
all-trans ROL 0.24 0.54 0.27 0.46
Kidney all-trans RE 0.48 0.17 0.28 0.31
all-trans ROL 0.69 0.57 0.86 1.76
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