The authors would like to acknowledge Satoshi Kinoshita and the University of Wisconsin (UW) Translational Research Initiatives in Pathology laboratory (TRIP) for preparing our tissues for light microscopy. This core is supported by the UW Department of Pathology and Laboratory Medicine, University of Wisconsin Carbone Cancer Center (P30 CA014520), and the Office of The Director-NIH (S10OD023526). Confocal microscopy was performed at the UW Biochemistry Optical Core, which was established with support from the UW Department of Biochemistry Endowment. This work was also supported by grants from the National Eye Institute (R01EY022086 to A. Ikeda; R01EY031748 to C. Bowes Rickman; P30EY016665 to the Department of Ophthalmology and Visual Sciences at the UW; P30EY005722 to the Duke Eye Center;NIH T32EY027721 to M. Landowski; F32EY032766 to M. Landowski), Timothy William Trout Chairmanship (A. Ikeda), FFB Free Family AMD Award (C. Bowes Rickman); and an unrestricted grant from the Research to Prevent Blindness (Duke Eye Center).

<ol>
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L., Johnson, L. V., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+models+and+therapies+for+age-related+macular+degeneration.">Cellular models and therapies for age-related macular degeneration.</a> Disease Models &amp; Mechanisms. 8 (5), 421-427 (2015).</li><li>Landowski, M., Bowes Rickman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+lipid+metabolism+for+the+treatment+of+age-related+macular+degeneration:+Insights+from+preclinical+mouse+models.">Targeting lipid metabolism for the treatment of age-related macular degeneration: Insights from preclinical mouse models.</a> Journal of Ocular Pharmacology and Therapeutics. 38 (1), 3-32 (2022).</li><li>Pennesi, M. E., Neuringer, M., Courtney, R. 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The authors of this protocol have no disclosures and conflicts of interest.

In this article, we introduced a phenotyping protocol for assessing the structural RPE pathologies of mouse models. We described the steps required for processing the eyes for various imaging techniques including light, transmission electron, and confocal microscopy, as well as the quantitation of typical pathologies observed via these imaging methods. We proved the effectiveness of our RPE phenotyping protocol by examining Tmem135 TG and 24-month-old WT mice, since these mice display RPE pathologies<su...

Age-related macular degeneration (AMD) is a common blinding disease that affects populations over the age of 551. Many researchers believe that dysfunction within the retinal pigmented epithelium (RPE) is an early and crucial pathobiological event in AMD2. The RPE is a monolayer of polarized cells tasked with maintaining the homeostasis of neighboring photoreceptors and choroidal blood vessels3. A variety of models exist to investigate disease-associated mechanisms within the RPE, including cell culture models4,5 and mice6,7,8. A recent report has described standardized protocols and quality control criteria for RPE cell culture models4, yet no report has attempted to standardize the phenotyping of the RPE in mouse models. In fact, many publications on mouse models of AMD lack a complete description of the RPE or quantification of the RPE pathologies in them. The overall goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using AMD mouse models.
Previous publications have noted the presence of several RPE pathologies in mice through three imaging techniques. For instance, light microscopy allows researchers to view the gross morphology of the murine retina (Figure 1A) and detect RPE pathologies such as RPE thinning, vacuolization, and migration. RPE thinning in an AMD mouse model is exemplified by a deviation in the RPE height from their respective controls (Figure 1B). RPE vacuolization can be divided into two separate categories: microvacuolization (Figure 1C) and macrovacuolization (Figure 1D). RPE microvacuolization is summarized by the presence of vacuoles in the RPE that do not affect its overall height, whereas macrovacuolization is indicated by the presence of vacuoles that protrude into the outer segments of the photoreceptors. RPE migration is distinguished by the focal aggregate of pigment above the RPE monolayer in a retinal cross-section (Figure 1E). It should be noted that migratory RPE cells in AMD donor eyes exhibit immunoreactivity to immune cell markers, such as cluster of differentiation 68 (CD68)9, and could represent immune cells engulfing RPE debris or RPE undergoing transdifferentiation9. Another imaging technique called transmission electron microscopy can permit researchers to visualize the ultrastructure of the RPE and its basement membrane (Figure 2A). This technique can identify the predominant sub-RPE deposit in mice, known as the basal laminar deposit (BLamD) (Figure 2B)10. Lastly, confocal microscopy can reveal the structure of RPE cells through imaging RPE flat mounts (Figure 3A). This method can uncover RPE dysmorphia, the deviation of the RPE from its classic honeycomb shape (Figure 3B). It can also detect RPE multinucleation, the presence of three or more nuclei within an RPE cell (Figure 3C). For a summary of the types of RPE pathologies present in current AMD mouse models, we refer researchers to these reviews from the literature6,7.
Researchers studying AMD should be aware of the advantages and disadvantages of using mice to investigate RPE pathologies prior to the phenotyping protocol. Mice are advantageous because of their relatively short life span and cost-effectiveness, as well as their genetic and pharmacologic manipulability. Mice also exhibit RPE degenerative changes, including RPE migration, dysmorphia, and multinucleation, that are observed in AMD donor eyes11,12,13,14,15,16,17; this suggests that similar mechanisms may underly the development of these RPE pathologies in mice and humans. However, there are key differences that limit the translatability of mouse studies to human AMD. First, mice do not have a macula, an anatomically distinct region of the human retina necessary for visual acuity that is preferentially affected in AMD. Second, some RPE pathologies in mice, like RPE thinning and vacuolization, are not typically seen in AMD donor eyes18. Third, mice do not develop drusen, a hallmark of AMD pathology19. Drusen are lipid- and protein-containing deposits with very few basement membrane proteins that form between the RPE basal lamina and the inner collagenous layer of Bruch&#39;s membrane (BrM)19. Drusen differ from BLamD, the common sub-RPE deposit in mice, in both their composition and anatomical location. BLamDs are age- and stress-dependent extracellular matrix-enriched abnormalities that form between the RPE basal lamina of BrM and the basal infoldings of the RPE20. Interestingly, BLamDs have a similar protein composition and appearance in both mice and humans6,10,21. Recent work suggests BLamDs may act in the pathobiology of AMD by influencing the progression of AMD to its later stages18,22; thus, these deposits may represent diseased RPE in the mouse retina. Knowledge of these benefits and limitations is critical for researchers interested in translating results from mouse studies to AMD.
In this protocol, we discuss the methods to prepare eyes for light, transmission electron, and confocal microscopy to visualize RPE pathologies. We also describe how to quantify RPE pathologies in an unbiased manner for statistical testing. As proof of concept, we utilize the RPE phenotyping protocol to investigate the structural RPE pathologies observed in transmembrane protein 135- (Tmem135) overexpressing mice and aged wild-type (WT) C57BL/6J mice. In summary, we aim to describe the phenotyping methodology to characterize the RPE in AMD mouse models, since there are currently no standard protocols available. Researchers interested in examining and quantifying pathologies of the photoreceptors or choroid, which are also affected in AMD mouse models, may not find this protocol useful for their studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 M Cacodylate Buffer pH7.2</td>
			<td>PolyScientiifc R&#38;D Company</td>
			<td>S1619</td>
			<td></td>
		</tr>
		<tr>
			<td>100 Capacity Slide Box</td>
			<td></td>
			<td></td>
			<td>Two are needed for this protocol (one for H&#38;E-stained slides and one for RPE flat mounts.)</td>
		</tr>
		<tr>
			<td>100% Ethanol&#160;</td>
			<td>MDS Warehouse</td>
			<td>2292-CASE</td>
			<td>Can be used to make diluted ethanol solutions in this protocol.</td>
		</tr>
		<tr>
			<td>1-Way Stopcock, 2 Female Luer Locks</td>
			<td>Qosina</td>
			<td>11069</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffer Solution (PBS)</td>
			<td></td>
			<td></td>
			<td>Premade 1x PBS can be used in this protocol.&#160;</td>
		</tr>
		<tr>
			<td>2.0 mL microtubes</td>
			<td>Genesee Scientific&#160;</td>
			<td>24-283-LR</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Cavity Embedding Capsule Substitute Mold</td>
			<td>Electron Microscopy Sciences</td>
			<td>70165</td>
			<td></td>
		</tr>
		<tr>
			<td>24 inch PVC Tubing with Luer Ends</td>
			<td>Fisher Scientific</td>
			<td>NC1376778</td>
			<td></td>
		</tr>
		<tr>
			<td>400 Mesh Gilder Thin Bar Square Mesh Grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>T400-Cu</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>MDS Warehouse</td>
			<td>2293-CASE</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent Underpads with Waterproof Moisture Barrier (23 inches by 24 inches)</td>
			<td>VWR</td>
			<td>56616-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable 237 ml&#160; Spray Bottle</td>
			<td>VWR</td>
			<td>23609-182</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor488 Conjugated Donkey anti-Rabbit IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Precision glide 19 Gauge Syringe Needle</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z192546</td>
			<td></td>
		</tr>
		<tr>
			<td>Bracken Forceps; Curved; Fine Cross Serrations; 4&#34; Length, 1 mm Tip Width</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5211</td>
			<td>Known as curved forceps in this protocol.</td>
		</tr>
		<tr>
			<td>Camel Hair Brush</td>
			<td>Electron Microscopy Sciences</td>
			<td>65575-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Dioxide Euthanasia Chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Dioxide Flow Meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Dioxide Tank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Castaloy Prong Extension Clamps</td>
			<td>Fisher Scientific&#160;</td>
			<td>05-769-7Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Cast-Iron L-shaped Base Support Stand</td>
			<td>Fisher Scientific&#160;</td>
			<td>11-474-207</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Prolifer Program</td>
			<td></td>
			<td></td>
			<td>Available to download: https://cellprofiler.org/releases</td>
		</tr>
		<tr>
			<td>Clear Nail Polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Colorfrost Microscope Slides Lavender</td>
			<td>VWR</td>
			<td>10118-956</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled H20</td>
			<td></td>
			<td></td>
			<td>Water from Milli-Q Purification System was used in this protocol.</td>
		</tr>
		<tr>
			<td>Dumont Thin Tip Tweezers; Pattern #55</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-4984</td>
			<td>Known as fine-tipped forceps in this protocol, and 3 are needed for this protocol (two for dissections and one for electron microscope processing).</td>
		</tr>
		<tr>
			<td>Electron Microscopy Grid Holder</td>
			<td>Electron Microscopy Sciences</td>
			<td>71147-01</td>
			<td></td>
		</tr>
		<tr>
			<td>EPON 815 Resin</td>
			<td>Electron Microscopy Sciences</td>
			<td>14910</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia Mark-It Tissue Marking Yellow Dye</td>
			<td>Fisher Scientific&#160;</td>
			<td>22050460</td>
			<td>Please follow manufacturer&#39;s protocol when using this tissue marking dye.&#160;</td>
		</tr>
		<tr>
			<td>Epredia Mounting Media</td>
			<td>Fisher Scientific</td>
			<td>22-110-610</td>
			<td>Use for mounting H&#38;E slides.&#160;</td>
		</tr>
		<tr>
			<td>Fiber-Lite Mi-150 Illuminator Series,150 w Halogen Light Source</td>
			<td>Dolan-Jenner Industries</td>
			<td>Mi-150</td>
			<td>Light source for dissecting microscope.</td>
		</tr>
		<tr>
			<td>Fiji ImageJ Program</td>
			<td></td>
			<td></td>
			<td>Available to download: https://imagej.net/downloads</td>
		</tr>
		<tr>
			<td>Flexaframe Castaloy Hook Connector</td>
			<td>Thermo Scientific&#160;&#160;</td>
			<td>14-666-18Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 2.5% in Phosphate Buffer, pH 7.4, 32%</td>
			<td>Electron Microscopy Sciences</td>
			<td>16537-05</td>
			<td></td>
		</tr>
		<tr>
			<td>JEM-1400 Transmission Electron Microscope (JEOL) with an ORIUS (1000) CCD Camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Benchtop Shaker</td>
			<td></td>
			<td></td>
			<td>Two are needed for these experiments. One should be at room temperature while the other should be in a 4 degree Celsius cold room.</td>
		</tr>
		<tr>
			<td>Laser Cryo Tag Labels</td>
			<td>Electron Microscopy Sciences</td>
			<td>77564-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Citrate</td>
			<td>Electron Microscopy Sciences</td>
			<td>17800</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica EM UC7Ultramicrotome</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Reichert Ultracut S Microtome</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LifterSlips</td>
			<td>Thermo Fisher Scientific</td>
			<td>22X22I24788001LS</td>
			<td>Use these coverslips for the RPE flat mounts as they have raised edges and accommodate the thickness of the RPE.</td>
		</tr>
		<tr>
			<td>Mayer&#39;s Hematoxylin</td>
			<td>VWR</td>
			<td>100504-406</td>
			<td></td>
		</tr>
		<tr>
			<td>McPherson-Vannas Micro Dissecting Spring Scissors</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5600</td>
			<td>Known as micro-dissecting scissors in protocol.&#160;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific&#160;</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td>Any AMD mouse model and its respective controls can work for this protocol.</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors; Standard Version; Curved; Sharp Points; 24 mm Blade Length; 4.5&#34; Overall Length</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5913</td>
			<td>Known as curved scissors in this protocol.</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube racks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1RS Confocal Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>SouthernBiotech</td>
			<td>0030-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Number 11 Sterile Disposable Scalpel Blades</td>
			<td>VWR</td>
			<td>21909-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 32%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>VWR&#160;</td>
			<td>21909-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Anti-ZO-1 Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>402200</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene Oxide</td>
			<td>Electron Microscopy Sciences</td>
			<td>20412</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blade</td>
			<td>VWR</td>
			<td>10040-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Shallow Tray for Mouse Perfusions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Eosin Y Alcoholic</td>
			<td>VWR</td>
			<td>89370-828</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharpie Ultra Fine Tip Black Permanent Marker</td>
			<td>Staples</td>
			<td>642736</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Rack for Staining</td>
			<td>Grainger</td>
			<td>49WF31</td>
			<td></td>
		</tr>
		<tr>
			<td>Squared Cover Glass Slips</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Dish with Cover</td>
			<td>Grainger</td>
			<td>49WF30</td>
			<td>Need 15 for H&#38;E staining procedure.</td>
		</tr>
		<tr>
			<td>Target All-Plastic Disposable Luer-Slip 50 mL Syringe&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>S7510-50</td>
			<td>Use only the syringe barrel.</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisher</td>
			<td>1464917</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Use for mounting RPE flat mounts.&#160;</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific&#160;</td>
			<td>22050283</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager 2 Light Microscope</td>
			<td></td>
			<td></td>
			<td>This microscope has the capacity to generate stitched 20x images. If a light microscope does not have this capacity, then take images of the entire retina that are slightly overlapping each other. Use Adobe Photoshop to stitch these images together. Please refer to the manuals of the Adobe Photoshop program for image stitching.&#160;</td>
		</tr>
		<tr>
			<td>Zeiss Stemi 2000 Dissecting Microscope</td>
			<td>Electron Microscopy Sciences</td>
			<td>65575-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to evaluate and quantify retinal pigmented epithelium pathologies in mouse models of age-related macular degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Completion of the RPE phenotyping protocol described in this article provides a quantitative analysis of the structural RPE abnormalities commonly observed in mouse models of AMD. To confirm the effectiveness of this protocol, we used it in mice that are known to display RPE pathologies, including transgenic mice that overexpress WT Tmem135 driven by the chicken beta-actin promoter (Tmem135 TG)30 and aged C57BL/6J mice31,32</su...

Watch this Scientific Journal Video about A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration at JoVE.com

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

a-protocol-to-evaluate-quantify-retinal-pigmented-epithelium

Research

JoVE Journal

Genetics

1.3K Views.  University of Wisconsin-Madison. Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy. 

Video: A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...