Name: cell-specific paired interrogation of the mouse ovarian epigenome and transcriptome
Uploaded: 2023-02-24T12:50:00
Description: Watch this Scientific Journal Video about Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome at JoVE.com

This work was supported by grants from the National Institutes of Health (NIH) (R01AG070035, R01AG069742, T32AG052363), BrightFocus Foundation (M2020207), and Presbyterian Health Foundation. This work was also supported in part by the MERIT award I01BX003906 and a Shared Equipment Evaluation Program (ShEEP) award ISIBX004797 from the United States (U.S.) Department of Veterans Affairs, Biomedical Laboratory Research and Development Service. The authors would also like to thank the Clinical Genomics Center (OMRF) and Imaging Core Facility (OMRF) for assistance and instrument usage.

All animal procedures were approved by the Institutional Animal Care and Use Committee at the Oklahoma Medical Research Foundation (OMRF). Parent mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred and housed under SPF conditions in a HEPA barrier environment on a 14 h/10 h light/dark cycle (lights on at 6:00am) at the OMRF.
NOTE: In this demonstration, we use a Cyp17iCre+/&#8722;(Strain # 028547, The Jackson Laboratory) male paired with a NuTRAP female (Strain...

<ol>
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The NuTRAP mouse model6&#160;is a powerful transgenic labeling approach for the paired interrogation of the transcriptome and epigenome from specific cell types that can be adapted to any cell type with an available Cre driver. Here, we demonstrate the specificity of the Cyp17-NuTRAP mouse model in targeting ovarian theca and stromal cells. The Cyp17-NuTRAP model can be used to further elucidate the theca and stromal cell-specific epigenetic mechanisms involved in ovarian aging, cancer, and diseas...

The ovaries are major players in somatic aging1, with distinct contributions from specific cell populations. The cellular heterogeneity of the ovary makes it difficult to interpret molecular results from bulk, whole-ovary assays. Understanding the role of specific cell populations in ovarian aging is key to identifying the molecular drivers responsible for fertility and health decline in aged women. Traditionally, the multi-omics assessment of specific ovarian cell types was achieved by techniques such as laser microdissection2, single-cell approaches3, or cell sorting4. However, microdissection can be expensive and difficult to perform, and cell sorting can alter cellular phenotypic profiles5.
A novel approach to assess ovarian cell-type-specific epigenomic and transcriptomic profiles uses the nuclear tagging and translating ribosome affinity purification (NuTRAP) mouse model. The NuTRAP model allows the isolation of cell-type-specific nucleic acids without the need for cell sorting by using the affinity purification methods: translating ribosome affinity purification (TRAP) and isolation of nuclei tagged in specific cell types (INTACT)6. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. By crossing the NuTRAP mouse with a cell-type-specific Cre line, the removal of the STOP cassette causes eGFP-tagging of the ribosomal complex and biotin/mCherry-tagging of the nucleus in a Cre-dependent manner6. The TRAP and INTACT techniques can then be used to isolate mRNA and nuclear DNA from the cell type of interest and proceed to transcriptomic and epigenomic analyses.
The NuTRAP model has been used in different tissues, such as adipose tissue6, brain tissue7,8,9, and the retina10, to reveal cell-type-specific epigenomic and transcriptomic changes that may not be detected in whole-tissue homogenate. The benefits of the NuTRAP approach over traditional cell sorting techniques include the following: 1) the prevention of ex vivo activational artifacts8, 2) the minimized need for specialized equipment (i.e., cell sorters), and 3) the increased throughput and decreased cost of cell-type-specific analyses. In addition, the ability to isolate cell-type-specific DNA and RNA from a single mouse allows for paired analyses that increase the statistical power. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes11,12,13, we targeted the NuTRAP expression system to stromal and theca cells using a Cyp17a1-Cre driver. Here, we demonstrate that the induction of the NuTRAP construct is specific to ovarian stromal and theca cells, and sufficient DNA and RNA for sequencing studies are obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with any available Cre line.
For the generation of a cell-type-specific ovarian NuTRAP mouse line, the nuclear tagging and translating ribosome affinity purification (NuTRAP) allele has a floxed STOP codon that controls the expression of BirA, biotin ligase recognition peptide (BLRP)-tagged mCherry/mRANGAP1, and eGFP/L10a. When crossed with a cell-type-specific Cre line, the expression of the NuTRAP cassette labels the nuclear protein mRANGAP1 with biotin/mCherry and ribosomal protein L10a with eGFP in a Cre-dependent manner. This allows for the isolation of nuclei and mRNA from specific cell types without the need for cell sorting. The NuTRAPflox/flox can be paired with a cell-type-specific Cre relevant to ovarian cell types to assess this.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 M Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>05292-1ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>Thermo Scientific</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>10% NP-40</td>
			<td>Thermo Scientific</td>
			<td>85124</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mg/mL Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C4859-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m cell strainer&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>All Prep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP antibody</td>
			<td>Abcam</td>
			<td>Ab290</td>
			<td>For TRAP and IHC (Rabbit polyclonal to GFP)</td>
		</tr>
		<tr>
			<td>Buffer RLT</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>RNA Lysis Buffer in protocol</td>
		</tr>
		<tr>
			<td>cOmplete, mini, EDTA-free protease inhibitor tablet</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>For TRAP Homogenization Buffer</td>
		</tr>
		<tr>
			<td>Cyp17iCre mouse model</td>
			<td>The Jackson Laboratory</td>
			<td>28547</td>
			<td>B6;SJL-Tg(Cyp17a1-icre)AJako/J</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Genotyping Primers</td>
			<td>IDT</td>
			<td>Custom</td>
			<td>Generic Cre - Jackson Laboratory protocol 22392, Primers: oIMR1084, oIMR1085, oIMR7338, oIMR7339</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Cyp17iCre - Jackson Laboratory protocol 30847, Primers: 21218, 31704, 31705, 35663</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>NuTRAP - Jackson Laboratory protocol 21509, Primers: 21306, 24493, 32625, 32626</td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor cocktail (100X)</td>
			<td>Thermo Scientific</td>
			<td>1861278</td>
			<td>For NPB Buffer</td>
		</tr>
		<tr>
			<td>M-280 Streptavidin Dynabeads&#160;</td>
			<td>Invitrogen</td>
			<td>11205D</td>
			<td>2.8 &#181;m bead diameter</td>
		</tr>
		<tr>
			<td>MixMate</td>
			<td>Eppendorf</td>
			<td>5353000529</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Kit: Nuclei EZ Prep</td>
			<td>Sigma-Aldrich</td>
			<td>Nuc101</td>
			<td>Contains Nuclei Lysis Buffer and Nuclei Storage Buffer</td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Thermo Scientific</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>2M KCl</td>
			<td>Thermo Scientific</td>
			<td>AM9640G</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Thermo Scientific</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EGTA</td>
			<td>Fisher Scientific</td>
			<td>50-255-956</td>
			<td></td>
		</tr>
		<tr>
			<td>NuTRAP mouse model</td>
			<td>The Jackson Laboratory</td>
			<td>29899</td>
			<td>B6;129S6-Gt(ROSA)26Sortm2(CAG-NuTRAP)Evdr/J</td>
		</tr>
		<tr>
			<td>Pierce DTT No-Weigh Format</td>
			<td>Thermo Scientific</td>
			<td>A39255</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Dynabeads</td>
			<td>ThermoFisher</td>
			<td>10004D</td>
			<td>For TRAP</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Invitrogen</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Heparin</td>
			<td>Fisher Scientific</td>
			<td>BP2425</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure 1M Tris-HCl, pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Tube Rotator</td>
			<td>Fisher Scientific</td>
			<td>NC9854190</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-specific paired interrogation of the mouse ovarian epigenome and transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

A schematic of the TRAP and INTACT protocols is shown in Figure 1. Here the specificity of the Cyp17-NuTRAP mouse model to ovarian stromal/theca cells is demonstrated by immunofluorescent imaging and RNA-Seq from TRAP-isolated RNA. First, immunofluorescence imaging of the eGFP signal in the ovary and localization of the eGFP signal to the theca and stromal cells were performed. Briefly, 5 &#181;m sections were deparaffinized with a xylene and ethanol gradient. For better eGFP signaling, the ...

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Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the ...

This method provides a technique to isolate nucleic acids from specific ovarian cell populations from the same mouse without the need for cell sorting. This technique can help identify how specific cell populations are changing under a variety of conditions. For example, with aging.The main advantage of this technique is that it allows for paired epigenomic and transcriptomic analysis from a specific ovarian cell type without the need for cell sorting. This significantly increases the throughput, decreases the cost, and eliminates the need for specialized equipment when performing cell type-specific analysis from ovarian tissue. Here, our goal is to identify the cell type-specific changes that occur with ovarian aging prior to reproductive senescence, which has implications in treating female infertility.This method can be applied to any cell type across body systems for which a cell type-specific Cre driver is available. The most difficult part of this technique is choosing an appropriate Cre line for your cell type of interest. Extensive validation of the cell specificity is recommended.To begin, collect one ovary from the euthanized mouse in 500 microliters of nuclei lysis buffer and chop the ovary into eight parts within the buffer using self-opening micro scissors. Transfer the minced ovary and the lysis buffer into a glass dounce homogenizer on ice and homogenize the ovary 10 to 20 times with the loose pestle A.Add 400 microliters of lysis buffer to the dounce homogenizer washing pestle A.And then homogenize the ovary with tight pestle B 10 to 20 times. Transfer the homogenate to a two-milliliter round bottom tube and centrifuge at 200 g for 1.5 minutes at four degrees Celsius to remove the undissociated tissue and blood vessels.After pre-wetting a 30-micrometer cell strainer with 100 microliters of lysis buffer, filter the supernatant through the strainer into a 15-milliliter conical tube. Transfer the nuclei containing mixture to a two-milliliter round bottom tube. Centrifuge the sample at 500 g for five minutes at four degrees Celsius and discard the supernatant.Resuspend the nuclei pellet in 250 microliters of ice cold lysis buffer by vortexing briefly at moderate to high speed. Bring the volume up to 1.75 milliliters by adding 1.5 milliliters of lysis buffer. Mix gently and rest the nuclei suspension on ice for five minutes.Pellet the nuclei by centrifugation at 500 g for five minutes at four degrees Celsius. And carefully discard the supernatant without disturbing the nuclei pellet. Resuspend the pellet in 200 microliters of ice cold storage buffer and vortex briefly to completely resuspend the nuclei pellet.Reserve 10%of the resuspended pellet as an input nuclei sample for downstream analysis. Take the resuspended nuclei pallet and make up the volume to two milliliters with ice cold nuclear purification buffer, or NPB. Add 30 microliters of resuspended beads for each sample into a two-milliliter round bottom tube.Then add one milliliter of NPB and resuspend well by pipetting. Place the tube on the magnetic rack for one minute to separate the beads. Discard the supernatant and remove the tube from the magnet.Resuspend the washed magnetic beads in one milliliter of NPB. Repeat the wash step for a total of three washes. And after the final wash, resuspend the beads in the initial volume of NPB.Add 30 microliters of the washed beads to two milliliters of the nuclear suspension and resuspend the beads nuclei mixture well by pipetting and gently inverting the tube. Place the tubes on a rotating mixer in a cold room or refrigerator for 30 minutes at low speed and incubate the input samples without beads in the same conditions. Place the tubes with the nuclei suspension and beads on the magnet for three minutes to separate the biotinylated nuclei bound to streptavidin beads from the negative fraction of nuclei.Remove the supernatant. Pass the negative fraction to a fresh two-milliliter tube and reserve it on ice. For each positive fraction tube, remove the tube from the magnet and resuspend the contents in one milliliter of NPB.Place the tube on the magnet for one minute and discard the supernatant. Resuspend the positive fraction bead nuclei mixture in 30 microliters of NPB. Transfer the ovary and 100 microliters of homogenization buffer into a glass dounce homogenizer and homogenize 10 to 20 times with the loose pestle A.Add 400 microliters of TRAP homogenization buffer to washing pestle A and transfer the homogenate to a two-milliliter round bottom tube.Wash the homogenizer with an additional one milliliter of TRAP homogenization buffer and transfer the washed volume to the two-milliliter round bottom tube. Centrifuge the homogenate at 12, 000 g for 10 minutes at four degrees Celsius. And transfer the cleared supernatant to a fresh two-milliliter round bottom tube.After discarding the pellet, transfer 100 microliters of the cleared homogenate to a fresh two-milliliter round bottom tube and reserve it as input on ice. Incubate the remainder of the cleared homogenate with five micrograms per milliliter anti-GFP antibody for one hour at four degrees Celsius while rotating in an end-over-end mixer. Incubate the input sample in the same condition.Transfer 50 microliters of the protein G magnetic beads for each sample into a two-milliliter round bottom tube. Add 500 microliters of low-salt wash buffer to the beads and pipette gently to mix. Place the tube into a magnetic stand for one minute to collect the beads against the side of the tube.After discarding the supernatant, remove the tube from the magnetic stand and add one milliliter of low-salt wash buffer to the tube. Pipette gently to mix. Again, place the tube into the magnetic stand and repeat the step for a total of three washes.After the final wash, resuspend the beads in the initial volume of low-salt wash buffer. Transfer the resuspended washed beads to the antigen sample antibody mixture and incubate at four degrees Celsius overnight while rotating in an end-over-end mixer. Incubate the input samples overnight in the equivalent conditions.After that, separate the positive fraction or the magnetic beads with the target ribosomes RNA from the negative fraction or the supernatant using the magnetic stand for 2.5 to 3 minutes. Aspirate the negative fraction. Place it in a fresh two-milliliter round bottom tube, and set it aside on ice.Add 500 microliters of high-salt wash buffer to the tube with the positive fraction and gently pipette to mix. Place the tube on a magnetic stand maintained on ice for one minute to separate the beads. After discarding the supernatant, remove the tube from the magnet and repeat the step for a total of three washes.After the final wash, resuspend the beads in 350 microliters of RNA lysis buffer supplemented with 3.5 microliters of 2-mercaptoethanol. Incubate the tubes at room temperature while mixing for 10 minutes at 900 RPM in a digital shaker. Separate the beads from the solution that now contains the target ribosomes RNA with a magnetic stand for one minute at room temperature.Collect the eluted positive fraction in a fresh 1.5 milliliter tube. Immunofluorescence imaging was performed on the paraffinized ovaries of Cyp17-Cre positive NuTRAP flox and Cyp17-Cre negative NuTRAP flox mice. EGFP protein was stained using goat anti-GFP primary antibody and Alexa 488 donkey anti-goat secondary antibody, and the nuclei was stained with DAPI.Images were taken on a fluorescence microscope at 20x magnification. The input and TRAP positive fraction RNA was isolated from Cyp17-Cre positive NuTRAP flox mouse ovaries and sequenced in a paired end manner. The principal component analysis of all the expressed genes showed a separation of the TRAP input and positive fractions in the first component.The volcano plot of differentially expressed genes between the input and positive fractions is shown here. The comparison of the TRAP positive fraction to the input showed an overall enrichment of the stroma/theca cell marker genes and the depletion of the oocyte, granulosa, immune, and smooth muscle cell marker genes in the TRAP positive fraction. After the first centrifugation of the nuclear suspension to remove undissociated blood vessels, the nuclei are in the supernatant.Make sure to keep the supernatant and discard the pellet at this point. Following the intact method, several epigenomic techniques can be conducted, including assays for chromatin accessibility or DNA modifications among others. Following the TRAP method, RT-qCPR or RNA sequencing can be conducted to assess the translatome.This is the first application of the NuTRAP method in the mouse ovary. This allows for high throughput epigenomic and transcriptomic analysis from specific ovarian cell types, which may be used to study ovarian aging or cancer.

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Research

JoVE Journal

Biology

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...