(A) IF staining for ABCA1 (yellow), ABCG1 (violet) and the RPE apical marker EZR (white) in retinas of 2-months-old wt mice. Lower panels show magnification of the RPE layer. Nuclei were …
(A) PCRs for Cre transgene, Abca1 floxed (flox) and excised (exc) alleles, and Abcg1 floxed and excised alleles were performed on genomic DNA extracted from eyecups and neural retinas of …
Abca1 (A), Abcg1 (B), Cre (C) and Mct3 (D) mRNA levels were measured by semi-quantitative real-time PCR in eyecups of 2-months-old Ctr and RPEΔAbca1;Abcg1 mice. Shown are data from individual …
(A) Fundus imaging of 2-months-old Ctr and RPEΔAbca1;Abcg1 mice showing altered pigmentation pattern in mutant mice. Corresponding retinal morphology analyzed by light (B) and electron (C) …
(A) RPE flat mounts from 4-months-old Ctr and RPEΔAbca1;Abcg1 mice were stained for ZO-1 (red) and β-cat (green). White arrowheads indicate loss of co-localization between ZO-1 and β-cat in mutant …
RPE cell Area and shape of RPE cells in RPE∆Abca1;Abcg1 and control mice.
Cre mRNA levels were measured by semi-quantitative real-time PCR in eyecups of 2- and 6-months-old RPEΔAbca1;Abcg1 and BEST1Cre mice. Shown are data from individual samples and means ± SD of the …
(A) Retinal morphology was analyzed by light microscopy in 2- and 6-months-old BEST1Cre animals. Yellow arrowhead indicates an example of occasionally observed minor alterations in the RPE. Retinal …
(A) Schematic representation of the vector packaged into AAV4 capsid in order to express Cre and GFP specifically in the RPE of Abca1flox/flox;Abcg1flox/flox mice. Length of the construct in base …
Lipid composition of eyecups (A), neural retinas (C) and plasma (D) from 2-months-old Ctr and mutant mice was measured by mass spectrometry-based methods. The following lipid classes were analyzed: …
Dark-adapted 2-months-old Ctr and RPEΔAbca1;Abcg1 mice were exposed to 5’000 lux for 10 min and the RHO content was measured in each retina. Dark controls were kept in darkness for the entire …
Rhodopsin regeneration in RPE∆Abca1;Abcg1 and control mice.
(A) Fundus images (upper panels) and OCT scans (lower panels, corresponding to red lines in fundus) of Ctr and RPEΔAbca1;Abcg1 mice at the indicated age. White arrowheads indicate sub-retinal …
(A) INL thickness was quantified from nasal-temporal panorama images at 6 months of age and presented as spidergram. Shown are means ± SD (N ≥ 3). Statistics: two-way ANOVA with Sidak’s multiple …
Scotopic and photopic ERGs were recorded with increasing light intensities from dark-adapted Ctr and RPEΔAbca1;Abcg1 mice at the indicated ages. Shown are mean ± SD (N = 3–6) of scotopic a- (A) and …
(A) RPE flat mounts from 4-months-old Ctr and RPEΔAbca1;Abcg1 mice were stained with phalloidin (green, staining actin filaments) and anti-IBA-1 (red). Shown are representative top-view images and …
2-months-old Ctr, double KO and single KO retinal sections were analyzed by light microscopy (A), ORO staining (B) and CRE IF (C). Single Abca1 mutant mice (RPEΔAbca1) showed an RPE phenotype …
LCLs derived from healthy individuals carrying the AMD decreased or increased risk ABCA1 genotypes were stimulated with an LXR agonist (1 μM) or DMSO vehicle control for 24 hr. (A) ABCA1 mRNA levels …
flox/-: detection of floxed and excised (KO) allele in ear biopsy.
Genotype | Name |
---|---|
Abca1flox/flox;Abcg1flox/flox | Cre-negative controls: Ctr |
Abca1flox/-;Abcg1flox/flox | |
Abca1flox/flox;Abcg1flox/- | |
Abca1flox/-;Abcg1flox/- | |
Abca1flox/flox;Abcg1flox/flox;BEST1Cre | RPE-specific double KOs: RPEΔAbca1;Abcg1 |
Abca1flox/-;Abcg1flox/flox;BEST1Cre | |
Abca1flox/flox;Abcg1flox/-;BEST1Cre | |
Abca1flox/-;Abcg1flox/-;BEST1Cre | |
Abca1flox/flox;Abcg1+/+;BEST1Cre | RPE-specific Abca1 single KOs: RPEΔAbca1 |
Abca1flox/-;Abcg1+/+;BEST1Cre | |
Abca1+/+;Abcg1flox/flox;BEST1Cre | RPE-specific Abcg1 single KOs: RPEΔAbcg1 |
Abca1+/+;Abcg1flox/-;BEST1Cre | |
Abca1+/+;Abcg1+/+;BEST1Cre | Cre-positive controls: BEST1Cre |
LCL | SNP | Genotype |
---|---|---|
Decreased risk (n = 3) | rs1883025 | TT |
rs2740488 | CC | |
Increased risk (n = 3) | rs1883025 | CC |
rs2740488 | AA |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Mus musculus) | Abca1 | NCBI gene ID: 11303 | ||
Gene (Mus musculus) | Abcg1 | NCBI gene ID: 11307 | ||
Strain, strain background (Mus musculus) | C57BL/6J (wt) | The Jackson Laboratory | RRID: IMSR_JAX:000664; The Jackson Laboratory: 000664 | |
Strain, strain background (Mus musculus) | BEST1Cre | Iacovelli et al., 2011 | RRID:IMSR_JAX:017557 | Name at the Jackson Laboratory: C57BL/6-Tg (BEST1-cre)1Jdun/J |
Strain, strain background (Mus musculus) | Abca1flox/flox; Abcg1flox/flox | The Jackson Laboratory | RRID:IMSR_JAX:021067 | Name at the Jackson Laboratory: B6.Cg-Abca1 tm1Jp Abcg1tm1Tall/J |
Antibody | anti-ABCA1 (rabbit polyclonal) | Novus Biologicals | RRID:AB_10000630; Novus Biologicals: NB400-105 | (1:250 for IF, 1:200 for WB) |
Antibody | anti-ABCG1 (rabbit monoclonal) | Abcam | RRID:AB_867471; Abcam: ab52617 | (1:100) |
Antibody | anti-EZR (mouse monoclonal) | Santa Cruz Biotechnology | RRID:AB_783303; Santa Cruz: sc-58758 | (1:500) |
Antibody | anti-CRE (rabbit polyclonal) | Merck | RRID:AB_10806983; Merck: 69050–3 | (1:300) |
Antibody | anti-IBA1 (rabbit polyclonal) | Wako Fujifilm | RRID:AB_839504; Wako Fujifilm: 019–19741 | (1:500) |
Antibody | anti-ZO1 (rabbit polyclonal) | Thermo Fisher Scientific | RRID:AB_2533456; Thermo Fisher Scientific: 40–2200 | (1:100) |
Antibody | anti-βcatenin (mouse monoclonal) | BD Biosciences | RRID:AB_397554; BD Biosciences: 610153 | (1:300) |
Antibody | anti-POU4F1 (mouse monoclonal) | Merck | RRID:AB_94166; Merck: MAB1585 | (1:100) |
Recombinant DNA reagent | pTR-BEST1-Cre-P2A-GFP (AAV vector plasmid) | This paper | Constructed from AAV plasmid materials at the University of Florida, laboratory of S. Boye | |
Sequence-based reagent | Random Primers | Promega | Promega: C1181 | |
Peptide, recombinant protein | Phalloidin-Alexa488 | Thermo Fisher Scientific | RRID:AB_2315147; Thermo Fisher Scientific: A12379 | (1:100) |
Commercial assay or kit | LipidTOX Red Neutral Lipid Stain | Thermo Fisher Scientific | Thermo Fisher Scientific: H34476 | (1:200) |
Commercial assay or kit | Protease Inhibitos Cocktail | Sigma-Aldrich | Sigma-Aldrich: P2417 | |
Commercial assay or kit | PowerUp Syber Green Master Mix | Thermo Fisher Scientific | Thermo Fishe rScientific: A25742 | |
Commercial assay or kit | NucleoSpin RNA isolation kit | Macherey-Nagel | Macherey-Nagel: 740949.250 | |
Chemical compound, drug | OilRedO (ORO) | Sigma-Aldrich | Sigma-Aldrich: O9755-25G | |
Chemical compound, drug | Oxalic Acid | Sigma-Aldrich | Sigma-Aldrich: 75688 | |
Chemical compound, drug | LXR agonist | Roche, Panday et al., 2006 | Roche: T0901317 | |
Chemical compound, drug | SPLASH | Avanti Polar Lipids | Avanti Polar Lipids: 330707 | |
Chemical compound, drug | d7-sphinganine (SPH d18:0) | Avanti Polar Lipids | Avanti Polar Lipids: 860658 | D-erythro-sphinganine-d7 |
Chemical compound, drug | d7-sphingosine (SPH d18:1) | Avanti Polar Lipids | Avanti Polar Lipids: 860657 | D-erythro-sphingosine-d7 |
Chemical compound, drug | Dihydroceramide (Cer d18:0/12:0) | Avanti Polar Lipids | Avanti Polar Lipids: 860635 | N-lauroyl-D-erythro -sphinganine |
Chemical compound, drug | Ceramide (Cer d18:1/12:0) | Avanti Polar Lipids | Avanti Polar Lipids: 860512 | N-lauroyl-D-erythro- sphingosine |
Chemical compound, drug | Glucosylceramide (GluCer d18:1/8:0) | Avanti Polar Lipids | Avanti Polar Lipids: 860540 | D-glucosyl-ß−1,1'-N -octanoyl-D-erythro -sphingosine |
Chemical compound, drug | Sphingomyelin (SM d18:1/12:0) | Avanti Polar Lipids | Avanti Polar Lipids: 860583 | N-lauroyl-D-erythro -sphingosylphosphorylcholine |
Chemical compound, drug | d7-sphingosine-1- phosphate (S1P d18:1) | Avanti Polar Lipids | Avanti Polar Lipids: 860659 | D-erythro-sphingosine -d7-1-phosphate |
Chemical compound, drug | Methanol | Honeywell | Honeywell: 34860 Riedel-de Haen | |
Chemical compound, drug | MTBE | Sigma-Aldrich | Sigma-Aldrich: 20256 | tert-Butyl methyl ether |
Chemical compound, drug | Chloroform | Sigma-Aldrich | Sigma-Aldrich: 650498 | |
Chemical compound, drug | Acetonitrile | Sigma-Aldrich | Sigma-Aldrich: 534851 | |
Chemical compound, drug | Isopropanol | Sigma-Aldrich | Sigma-Aldrich: 59300 | |
Software, algorithm | ImageJ Tissue Cell Geometry macro | Institute for Research in Biomedicine, Barcelona, Spain | http://adm.irbbarcelona.org/image-j-fiji | |
Software, algorithm | Relative Quantification Software | Thermo Fisher Cloud | https://www.thermofisher.com/uk/en/home/digital-science/thermo-fisher-connect/all-analysis-modules.html | |
Software, algorithm | GraphPad Prism, version 7 | GraphPad | RRID:SCR_002798 | |
Software, algorithm | Tracefinder Clinical 4.1 | Thermo Fisher Scientific | ||
Other | transcend TLX I eluting pump | Thermo Fisher Scientific | ||
Other | Q-Exactive | Thermo Fisher Scientific | ||
Other | Mini-PROTEAN Precast Gels, 4–15% polyacrylamide | BioRad | BioRad: 4561086DC | |
Other | C30 Accucore LC column | Thermo Fisher Scientific | Thermo Fisher Scientific: 7826–152130 | 150 mm * 2.1 mm * 2.6 µm |
Supplementary tables.
(A) Absolute concentrations of analyzed lipid classes from 2-months-old Ctr and RPEΔAbca1;Abcg1 mice. (B) RHO measurements from 2-months-old Ctr and RPEΔAbca1;Abcg1 neural retinas. (C) Primers used for genotyping. (D) Primers used for gene expression analysis.