Name: isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders
Uploaded: 2016-12-05T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM33397. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.G.G. is American Cancer Society Professor of Developmental Genetics.

General information about frogs and salamanders, as well as sources of animals, can be found at the following websites: Xenbase (http://www.xenbase.org) and Sal-Site (http://www.ambystoma.org). This protocol follows the animal care guidelines of the Department of Embryology of the Carnegie Institution for Science.
1. Solutions
<ol>
	<li>Make 10 L &#34;frog water&#34;: Add 10 mL of 1 M CaCl2 and 10 mL of 1 M NaHCO3 to 10 L deionized or dechlorinated H2O with s...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octagonal+nuclear+pores.">Octagonal nuclear pores.</a> J Cell Biol. 32, 391-399 (1967).</li><li>L&#246;schberger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+visualizes+the+eightfold+symmetry+of+gp210+proteins+around+the+nuclear+pore+complex+and+resolves+the+central+channel+with+nanometer+resolution.">Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution.</a> J Cell Sci. 125, 570-575 (2012).</li><li>Gottfert, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coaligned+dual-channel+STED+nanoscopy+and+molecular+diffusion+analysis+at+20+nm+resolution.">Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.</a> Biophys J. 105, L01-L03 (2013).</li><li>Wallace, R. A., Jared, D. W., Dumont, J. N., Sega, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+incorporation+by+isolated+amphibian+oocytes:+III.+Optimum+incubation+conditions.">Protein incorporation by isolated amphibian oocytes: III. Optimum incubation conditions.</a> J. Exp. Zool. 184, 321-333 (1973).</li><li>Bridger, J., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situhybridization+to+DNA.">Fluorescence in situhybridization to DNA.</a> Cells: A Laboratory Manual. 3, 111.111-111.136 (1998).</li><li>Singer, R. H., Spector, D. L., Goldman, R. D., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+to+RNA.">In situ hybridization to RNA.</a> Cells: A Laboratory Manual. 3, 111-116 (1998).</li><li>Gardner, E. J., Nizami, Z. F., Talbot, C. C., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+intronic+sequence+RNA+(sisRNA),+a+new+class+of+noncoding+RNA+from+the+oocyte+nucleus+of+Xenopus+tropicalis.">Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis.</a> Genes Dev. 26, 2550-2559 (2012).</li><li>Talhouarne, G. J., Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lariat+intronic+RNAs+in+the+cytoplasm+of+Xenopus+tropicalis+oocytes.">Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.</a> RNA. 20, 1476-1487 (2014).</li><li>Duryee, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+nuclei+and+non-mitotic+chromosome+pairs+from+frog+eggs.">Isolation of nuclei and non-mitotic chromosome pairs from frog eggs.</a> Arch. Exp. Zellforsch. 19, 171-176 (1937).</li></ol>

The first observations of &#34;living&#34; LBCs from hand-isolated GVs of frogs and salamanders were made nearly 80 years ago by the American biologist William Duryee,19 before the introduction of phase contrast and DIC microscopy, before fluorescent immunostaining, and before FISH. The advantages of LBCs for investigating details of chromosome structure and transcription at the individual gene level required development of techniques to attach the LBCs to glass slides, to fix them in a lifelike manner, and to...

Most vertebrates, with the notable exception of marsupials and placental mammals, produce large yolky eggs. Despite their sometimes enormous size, these eggs are single cells that reach their final dimension while still in the ovary of the female. Ovarian eggs are called oocytes and each typically contains a single giant nucleus, known since the early 19th century as the germinal vesicle or simply GV.1 Oocytes of the common laboratory frogs, Xenopus laevis and Xenopus tropicalis, reach a maximal diameter of 1.2 mm and 0.8 mm respectively (Figure 1). The GVs from mature oocytes of these two frogs are 0.3 - 0.4 mm in diameter (Figures 2, 3). Salamanders typically have even larger oocytes and GVs. Fully mature oocytes of the Mexican axolotl, Ambystoma mexicanum, are more than 2 mm in diameter and the GV is about 0.5 mm. Thus, these nuclei are readily visible to the naked eye and can be manipulated in many ways that are impossible with the nuclei of typical somatic cells.
Equally remarkable is the gigantic size of the chromosomes within the GV, a fact recognized already at the end of the 19th century. Individual chromosomes of Ambystoma and other salamanders can be up to 1 mm in length (Figures 4, 5). Those of Xenopus are considerable smaller, although with lengths up to 100 &#181;m or more, they dwarf the typical somatic chromosomes of most organisms. An important feature of oocyte chromosomes is their extraordinary transcriptional activity, which leads to one of their most characteristic morphological features &#8212; hundreds of paired lateral loops (Figure 5). Each loop consists of one or a few transcription units that actively synthesize RNA. The loops give oocyte chromosomes a fuzzy appearance, which led to the name &#34;lampbrush&#34; chromosome after their superficial resemblance to the brushes used in earlier times to clean kerosene lamp chimneys.2
The focus of this paper is on the use of isolated GVs to study LBCs and nuclear organelles (nucleoli, histone locus bodies, and speckles). Two rather different techniques will be described. In the first, more common technique, GVs are isolated in a saline solution using jeweler&#39;s forceps, briefly rinsed to remove adherent yolk, and the nuclear envelope is removed, again with jeweler&#39;s forceps. The gelatinous contents, containing the LBCs and nuclear organelles, are allowed to settle onto a glass microscope slide or coverslip. Such preparations can be examined directly by phase contrast or DIC microscopy. Alternatively, preparations may be centrifuged to attach the LBCs and organelles to the slide or coverslip. Such preparations can then be processed for detailed molecular analysis of nucleic acids and proteins, primarily by immunofluorescence and fluorescent in situ hybridization (FISH).3-7
A second technique involves isolation of the GV in mineral oil.8 Oil-isolated GVs remain transcriptionally active for many hours and are potentially useful for studies where one wants the nuclear contents to be as lifelike as possible.9,10 Because the refractive index of the nuclear &#34;sap&#34; is close to that of the LBCs and other nuclear organelles (Figure 3), microscopical techniques can be a challenge with oil-isolated GVs.
Finally, because of their size and ease of manipulation, GVs are ideal material for studies on the nuclear envelope. The nuclear pore complex was first described from electron microscopic studies on amphibian GV envelopes11 and more recent superresolution observations have used the same material.12,13

<table border="0" cellpadding="0" cellspacing="0" width="1562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Paraformaldehyde (reagent grade, crystalline)</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:213px;">P6148-500G</td>
			<td style="width:643px;">Despite warnings in many protocols, a concentrated solution can be stored indefinitely at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>A5040-100G</td>
			<td>Sometimes referred to as MS-222</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethicon RB-1 1/2 circle taper point 3-0 sutures</td>
			<td>VWR&#160;</td>
			<td>95057-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraplast (paraffin wax)</td>
			<td>Sigma-Aldrich</td>
			<td>P3558-1KG</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-Phenylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>P6001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Grocery Store</td>
			<td></td>
			<td>Commercial Knox gelatin works fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Seal cover glass&#160; 22 x 22 mm #1 1/2 (0.16-0.19 mm thick)</td>
			<td>Electron Microscopy Sciences</td>
			<td>63786-01</td>
			<td>These coverslips are the recommended thickness for superresolution microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps #5</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td>http://www.emsdiasum.com/microscopy/products/tweezers/dumont_positive_action.aspx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraffin oil (light)&#160;</td>
			<td>EMD Chemicals</td>
			<td>PX0047-1</td>
			<td>For isolating GVs in oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive in situ PCR and hybridization chambers (25 &#181;l).</td>
			<td>BioRad</td>
			<td>Frame-Seal Slide Chambers #SLF0201</td>
			<td>http://www.bio-rad.com/en-us/sku/slf0201-frame-seal-slide-chambers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone isolators</td>
			<td>Grace-Biolabs</td>
			<td>select from catalog link</td>
			<td>http://www.gracebio.com/life-science-products/microfluidics/silicone-isolators.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coplin jars and staining dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>select from catalog link</td>
			<td>http://www.emsdiasum.com/microscopy/products/histology/staining.aspx</td>
		</tr>
	</tbody>
</table>

isolation of giant lampbrush chromosomes from living oocytes of frogs and salamanders

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and salamanders. Individual LBCs can be up to 1 mm in length and they reside in a gigantic nucleus, itself up to 0.5 mm in diameter. The large size of the chromosomes permits unparalleled observations of active genes by light optical microscopy, but at the same time special techniques are required for isolating the nucleus, removing the nuclear envelope, and spreading the chromosomes on a microscope slide. The oocyte nucleus, also called the germinal vesicle (GV), is isolated in a medium that allows partial gelling of the nuclear actin and preserves the delicate structure of the LBCs. This step is carried out manually under a dissecting microscope using jeweler&#39;s forceps. Next, the nuclear envelope is removed, again manually with jeweler&#39;s forceps. The nuclear contents are quickly transferred to a medium that disperses the actin gel and allows the undamaged LBCs to settle onto a microscope slide. At this point the LBCs and other nuclear organelles can be viewed by phase contrast or differential interference contrast microscopy, although finer details are obscured by Brownian motion. For high resolution microscopical observation or molecular analysis, the whole preparation is centrifuged to attach the delicate LBCs firmly to the slide. A brief fixation in paraformaldehyde is then followed by immunofluorescent staining or in situ hybridization. LBCs are in a transcriptionally active state and their enormous size permits molecular analysis at the individual gene level using confocal or super-resolution microscopy.

To examine giant lampbrush chromosomes one begins by isolating oocytes from a frog or salamander. Figure 1 shows a group of mature oocytes in a buffered saline solution after removal from the ovary of the frog, Xenopus. Such oocytes remain in good condition for days at room temperature. The nucleus (or germinal vesicle) is then removed from an oocyte with jeweler&#39;s forceps, either in a saline solution (Figure 2) or in oil (Figure 3</s...

Watch this Scientific Journal Video about Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders at JoVE.com

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

We present simple techniques for isolating giant transcriptionally active lampbrush chromosomes from living oocytes of frogs and salamanders. We describe how to observe these chromosomes &#34;alive&#34; by phase contrast or differential interference contrast, and how to fix them for fluorescent in situ hybridization or immunofluorescent staining.

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and ...

The overall goal of this procedure is to isolate the giant nucleus, or germinal vesicle, from an amphibian oocyte, and to spread the lampbrush chromosomes, and other nuclear organelles, on a microscope slide for immunostaining, in citu hybridization, and other molecular techniques. This method can help answer key questions about the basic structure of chromosomes, nucleoli, and other nuclear organelles as well as molecular details of transcription, splicing, and RNA export. The main advantage of this technique is the ease with which chromosome structure and transcription can be examined by a conventional light microscopy, using immunofluorescence, in citu hybridization, and other molecular techniques.Demonstrating the procedure will be Dr.Zehra Nizami, a postdoc from my laboratory. After preparing all of the required solutions, assemble well slides, composed of subbed, glass slides, to which plastic wells have been attached with paraffin, as discussed in the text protocol. Then gather ovarian tissue previously collected from an adult, female frog or a salamander, and fill a small Petri dish with oocyte culture medium.Proceed to place a portion of the isolated tissue, one containing approximately 10 to 20 oocytes, into this plate. Next, identify a single oocyte and use two pairs of jewelers forceps to sever the stalk that connects it to the ovary wall. Once it is freed, transfer the oocyte to a new Petri dish containing germinal vesicle, or GV, isolation medium.Under low magnification, locate the animal pole of the oocyte, which can be identified by its dark coloration. Then use forceps to poke a large hole near the animal pole and proceed to gently squeeze out the transparent oocyte nucleus, also referred to as the GV.Afterwards, roll the GV away from any yolk that was also extruded. To remove yolk that remains tightly adhered, draw the nucleus in and out of a pipette with a tip having a diameter slightly larger than that of the GV.Following the collection of a frog GV, immediately transfer it to dispersal medium in order to prevent nuclear hardening that would interfere with subsequent steps.Next, with two pairs of forceps, grasp the nuclear envelope near the top of the GV, taking care not to press down. While still gripping the envelope, proceed to pull the two forceps apart and expect the contents of the nucleus to pop out. To collect these contents, first prepare an adjustable 20 microliter pipette with a cut tip as described in the text protocol.Then, from the dish, proceed to draw up five microliters of dispersal liquid followed by five microliters of the solution with the GV contents. Once these contents have been collected, transfer them to a well slide filled with dispersal solution. Ensure that the liquid in the slide has a convex surface so as to avoid the trapping of bubbles in subsequent steps.Afterwards, position an 18 millimeter cover slip over the well and place the assembled slide into a moist chamber for 10 to 60 minutes. During this time, expect the nuclear gel to slowly disperse, so that the chromosomes and nuclear organelles come to lie on the surface of the glass. After removing a nucleus from a salamander oocyte, leave it in the isolation medium.Note that salamander GVs, unlike those of the frog species used here, will not unduly harden in this solution. Remove the nuclear envelope from the salamander GV using the same forceps-based technique as described for frogs. Next, pick up the slightly gelled nuclear contents with a pipette having a cut tip, and transfer the gel to a Petri dish containing dispersal solution.Quickly rinse the pipette in the dispersal solution, and use it to collect the nuclear gel. Then introduce the gel into a well slide, add a cover slip, and allow the gel to disperse as previously described. Then use phase-contrast to view the nuclear contents, and identify the lampbrush chromosomes, abbreviated as LBCs, by their large size and paired, lateral loops.After confirming that the LBCs are unbroken, prepare the slide for centrifugation by placing a piece of filter paper on the cover slip. Then press down to remove excess liquid from the chamber. Next, add petroleum jelly to each edge of the cover slip.Heat a metal rod to approximately 70 to 80 degrees Celsius and use it to melt the jelly and seal the cover slip onto the underlying plastic. Repeat this procedure for other slides with LBCs of good quality. Once all the cover slips have been affixed, position the slides in carriers for centrifugation and ensure that opposite carriers are balanced.Using the slow start function, centrifuge the slides as described in the text using custom-built carriers. Alternatively, a standard tabletop centrifuge can be used where slides are taped to a plate and spun. To prepare the nuclear contents for immunostaining or in citu hybridization, first wipe away the temporary Vaseline seal with a razor blade, and then place the slide into the rack of a standard slide-staining dish.Next, add enough paraformaldehyde to cover the slides. Upon submergence, use blunt forceps to remove and discard each cover slip. Leave the slides in fixative for 15 to 30 minutes.Following fixation, place a slide into a staining dish containing ice cold PBS. Then insert a sharp razor blade at the edge of the plastic square, and pry this plastic well loose. Once this process has been repeated for all the slides, proceed with staining as discussed in the text protocol.Shown in this dark-field illumination image are the nuclear contents from a single axolotl GV in prophase of the first meiotic division, collected and prepared using the described protocol. 14 large, paired LBCs are apparent, as are amplified nucleoli. Additional staining of this preparation, with an antibody against phosporylted RNA polymerase II, shown here in green, reveals the locations of histone locus bodies and also provides insight into the robust, transcriptional activity of these oocyte chromosomes.Higher magnification of a single axolotl LBC, in this instance one that has been stained green for RNA polymerase and blue for DNA, demonstrates the most unique morphological feature of these chromosomes. Hundreds of paired lateral loops, each of which represents at least one transcription unit that actively synthesizes RNA. Comparison with a similarly stained frog LBC, imaged at the same magnification, emphasizes the enormous size difference between frog and salamander chromosomes, a difference that correlates with the total DNA content of the genomes of the species used.However, even in the frog chromosome, the individual transcription units can be seen as loops of chromatin projecting laterally from the chromosome axis, as shown in this higher magnification image. Oocytes of fish, birds, reptiles, and some invertebrates also contain giant lampbrush chromosomes. Remarkably, lampbrush chromosomes are generated when sperm heads from other organisms, including humans, are injected into the amphibian germinal vesicle.Once the technique is mastered, a batch of AGV spreads can be prepared in one hour, and then centrifuged and fixed within another one to two hours. After watching this video, you should have a good understanding of how to isolate a GV and spread the lampbrush chromosomes for further study using molecular probes.

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The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

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), ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...