Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Plate 52 Whole-mount preparation of rat retina in vivo electroporated at P0 with CAG-GFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the CAG (chicken β-actin promoter

with cytomegalovirus enhancer) promoter and harvested at P21. Images are from the scleral side. Bright-field (A), GFP (B), and merged (C ) images are shown. (See figure 51.3.)

Plate 53 In vivo electroporated retina (P0 electroporation, section). Rat retinas were in vivo electroporated with CAG-GFP at P0 and harvested at P2 (top panel) or P20 (bottom panel). At P2, most of the GFP-positive cells have the morphology of progenitor/precursor cells, suggesting that DNAs are preferentially transfected to progenitor/precursor cells. Retinogenesis is completed within the

first 2 weeks after birth. At P20, GFP is observed in four differentiated cell types: rod photoreceptors, bipolar cells, amacrine cells, and Müller glia. Early-born cell types (cone, horizontal, and ganglion cells) are not labeled by P0 electroporation. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; VZ, ventricular zone. (See figure 51.4.)

Plate 54 In vivo electroporated retina (E14 electroporation, section). Mouse embryonic retinas were electroporated with UBGFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the human ubiquitin promoter, at E14 in utero, and harvested at P20. Early-born cell types (cone, amacrine, horizontal, and ganglion cells) are clearly labeled with GFP, while late-born cell types (bipolar and Müller glial cells), which are generated from E14

RPCs after several rounds of cell division, are poorly labeled. This is probably due to dilution of introduced plasmids. Red arrowheads indicate the labeled cone photoreceptors. Yellow arrowhead indicates the labeled horizontal cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. (See figure 51.6.)

Plate 56 In vitro electroporated retinal explant (whole mount). Mouse retinas of P0 CD1 (A and B), adult CD1 (C and D), or adult Swiss Webster mice with a retinal degeneration mutation (E and F) were in vitro electroporated with CAG-GFP from the scleral side (A, C, and E) or from the vitreal side (B, D, and F) and cultured for

5 days. Images A, C, and E are from the scleral side and images B, D, and F are from the vitreal side. Note that only the scleral side of developing retina or of degenerated retina is highly transfectable. In E, most of the GFP-positive cells are Müller glial cells. (See figure 51.8.)

Plate 57 Temporal regulation of gene expression in the retina using inducible Cre recombinases. A, CAG-CreERT2: Fusion protein (CreERT2) between Cre recombinase and the mutated ligand-binding domain (ERT2) of the human estrogen receptor is expressed under the control of the CAG promoter. CAG-ERT2 CreERT2: Fusion protein (ERT2CreERT2) composed of Cre and two ERT2 domains is expressed under the control of the CAG promoter. CreERT2 and ERT2CreERT2 are conditionally activated in response to 4-OHT. B, CALNL-DsRed: Cre/loxP-dependent inducible expression vector. DsRed is expressed only in the presence of Cre. C, A scheme of the experiment. D–I, P0 rat retinas were coelectroporated with three plasmids: CAG-GFP (transfection control),

CALNL-DsRed (recombination indicator), and CAG-CreERT2 (D) or CAG-ERT2CreERT2 (E and F). The retinas were stimulated without 4-OHT (D and E) or with 4-OHT (F) by IP injection at P20 and then harvested at P21. Whole-mount preparations of the harvested retinas are shown. G–I, Sections of the retinas shown in D–F. Cell nuclei were stained with DAPI. When CreERT2 was used, significant background recombination was observed even in the absence of 4-OHT. On the other hand, ERT2CreERT2 had no detectable basal activity in the absence of 4-OHT. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (See figure 51.9.)

Plate 58 Spatial regulation of gene expression in the retina using cell type–specific promoters. Retinal cell type–specific promoters were fused to DsRed cDNA and electroporated into P0 rat retinas. The retinas were harvested at P20 (A–F and H) or P2 (G), sectioned,

and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), and Hes1 (G and H) were used to express DsRed. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (See figure 51.10.)

Plate 59 Multicolor labeling of the retina using cell type–specific promoters. P0 rat retina was electroporated with three reporter constructs: rhodopsin promoter 2.2K-CFP (specific for rods), Cabp5 promoter 4.7K-YFP (specific for a subset of bipolar cells),

and Cralbp promoter 4.0K-DsRed (specific for Müller glia). The retina was harvested at P20, sectioned, and stained with DAPI. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. (See figure 51.11.)

Plate 60 Lineage tracing experiments in the retina using the Cre/loxP system and cell type–specific promoters. P0 rat retinas were coelectroporated with retinal cell type–specific promoter Cre and CALNL-DsRed (recombination indicator). DsRed expression, induced by Cre/loxP-mediated recombination, was driven by the ubiquitous CAG promoter. The retinas were harvested at P20,

sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), Hes1 (G), and Rax (H) were used to express Cre. Yellow arrowheads indicate the labeled rods. Blue arrowhead indicates the labeled bipolar cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (See figure 51.12.)

Plate 61 Inducible expression in the differentiated Müller glia (A). Clusterin promoter was fused to ERT2CreERT2 cDNA and coelectroporated into P0 rat retinas with CALNL-DsRed (recombination indicator) and CAG-GFP (transfection control). Retinas were stimulated with or without 4-OHT at P14 by IP injection, then harvested at P16. B, Whole-mount preparation of the trans-

fected retina harvested at P16 without 4-OHT stimulation. No DsRed expression was detected. C, Whole-mount preparation of the transfected retina stimulated with 4-OHT at P14 and harvested at P16. D, The retina shown in C was sectioned and stained with DAPI. Only Müller glial cells are labeled with DsRed. (See figure 51.13.)

Plate 62 Inducible RNAi in the retina. A, CAG-mir30: The mir30 expression cassette is expressed under the control of the CAG promoter. The mir30 expression cassette has the hairpin stem composed of siRNA sense and antisense strands (22nt each), a loop derived from human mir30 (19nt), and 125nt mir30 flanking sequences on both sides of the hairpin. The mir30 primary transcript is processed to generate the mature shRNA. CALSLmir30: Cre-dependent inducible shRNA expression vector carrying a floxed transcriptional stop cassette (3xpolyA signal sequences).

Only in the presence of Cre, the mir30 expression cassette is expressed under the control of the CAG promoter. B and C, Conditional GFP knockdown in the retina. CAG-GFP and CAGDsRed and CALSL-mir30(GFPshRNA) expressing an shRNA against GFP were coelectroporated without (B) or with (C ) the rhodopsin promoter Cre into P0 rat retinas. The retinas were harvested at P20, sectioned, and stained with DAPI. Rod-specific GFP knockdown was observed in the presence of the rhodopsin promoter Cre. (See figure 51.14.)

Plate 63 A, A single rod photoreceptor isolated from adult wt-Gfp mice shows the expression of GFP. B, The same cell shows staining for rhodpsin (red) in the outer segment (OS) and inner segment (IS) regions and bis-benzimide staining (blue) for the nucleus.

C, Samples of dissociated cells are viewed under the microscope before (shown) and after flow sorting. D, Flow cytometry allows the GFP-positive population of cells to be gated (R3) and sorted separately. (See figure 55.1.)

Plate 64 Top, The retina is a complex, striated tissue with highly organized neuronal layers. The inner retinal vasculature forms three distinct plexuses: The superficial plexus forms within the ganglion cell layer (GCL), the intermediate plexus forms at the inner edge of the inner nuclear layer (INL), and the deep plexus

forms at the outer edge of the INL. Bottom, Posterior to the retina are the RPE, Bruch’s membrane, and the choriocapillaris. The RPE performs many functions that are critical to retinal function. (See figure 56.1.) (Top, From Dorrell and Friedlander, 2006. Bottom, From Strauss, 2005.)