The authors acknowledge Marjo van Brakel, Dominique Smeets, Guillaume van de Zande, Patricia van Cleef and Milan Intezar for their expertise and technical assistance. Funding sources: Merck Serono (A16-1395), Goodlife, and Ferring.

The TurnerFertility study protocol has been approved by the Central Committee on Research Involving Human Subjects (NL57738.000.16). In this study, the ovarian cortex tissue of 93 females with TS was obtained. Materials that require safety precautions are listed in Table 1.
Table 1: Safety precautions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td colspan="3">Hazard</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td colspan="3">Severe skin burns and irritation of the respiratory system</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td colspan="3">Highly flammable</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td colspan="3">Toxic after inhalation, ingestion and skin contact</td>
		</tr>
		<tr>
			<td>Formamide 
			(in fluorescence probes)</td>
			<td colspan="3">May harm the unborn child</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td colspan="3">Highly flammable, toxic by inhalation, ingestion and skin contact</td>
		</tr>
		<tr>
			<td>Nonidet P40</td>
			<td colspan="3">Irritating to the skin or eyes</td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td colspan="3">Breathing difficulties after inhalation</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td colspan="3">Highly flammable, toxic after inhalation and skin contact. Avoid contact with the eyes.</td>
		</tr>
	</tbody>
</table>
Table 1: Materials that require safety precautions.
1. FISH on isolated individual ovarian cortex cells
<ol>
	<li>Dissociation of ovarian cortex tissue to obtain individual cells
	<ol>
		<li>Cut the cryopreserved/thawed ovarian cortex tissue into small pieces of approximately 1 mm x 1 mm x 1 mm using a scalpel.</li>
		<li>Enzymatically digest the tissue fragments in 4 mL of pre-warmed (37 &#176;C) L15 medium containing 0.1 mg/mL tissue dissociation enzyme mix, 10 &#181;g/mL DNase I, and 1 mg/mL collagenase I from C. histolyticum for a maximum of 75 min at 37 &#176;C. Pipet the digestion mix up and down every 15 min.</li>
		<li>Stop the enzymatic digestion by adding 4 mL of cold L15 supplemented with 10% fetal bovine serum (FBS). Wash the dissociated tissue once with 8 mL of cold L15 medium by centrifugation at 500 x g and resuspend in 500 &#181;L of L15 medium without vortexing to avoid damage to the cells.</li>
		<li>Transfer the cell suspension containing largely individual stromal cells and small follicles (oocytes surrounded by a single layer of granulosa cells) to a plastic Petri dish and examine the cell suspension under a stereomicroscope (100x magnification).</li>
		<li>Pick up the small follicles (&#60;50 &#181;m) manually by using a 75 &#181;m plastic pipette and transfer the follicles to a droplet of L15 medium supplemented with 10% FBS at 4 &#176;C to prevent the aggregation of follicles. Perform follicle pick-up for a maximum of 30 min. To improve follicular cell spreading prior to FISH analysis, transfer the purified follicles to a solution of 0.06% trypsin, 1 mg/mL ethylenediaminetetraacetic acid (EDTA), and 1 mg/mL glucose and incubate for 20 min at 37 &#176;C.</li>
		<li>Obtain ovarian stromal cells from the cortex cell suspension using a 75 &#181;m plastic pipette, taking special care to avoid contamination with small follicles.</li>
	</ol>
	</li>
	<li>FISH analysis of individual ovarian cell
	<ol>
		<li>Transfer the treated ovarian follicles (recommended n = 5-20) with trypsin/EDTA/glucose or stromal cells (n &#62; 1,000) to droplets of 5 &#181;L of 0.15 mM KCl/15 &#181;L of Dulbecco&#39;s phosphate buffered saline (DPBS) on a slide and incubate for 20 min at 37 &#176;C.</li>
		<li>Dry and pre-fixate the slides in 300 &#181;L of 0.05 mM KCl/7.5% acetic acid/22.5% methanol for 2 min at room temperature (RT). Cover the slides with methanol/acetic acid (3:1) for 2 min at RT to finalize fixation.</li>
		<li>Make 20x standard sodium citrate (SSC) by adding 876 g of sodium chloride and 441 g of tri-sodium citrate dihydrate in 5 L of distilled water. Next, add 100 mL of the 20x SSC to 900 mL of demineralized (demi) water to obtain 2x SSC. Wash the sample in 2x SSC at 73 &#176;C, cover it with 100 &#181;L of 2% proteinase K, and seal it with a coverslip. Incubate the slides for 10 min at 37 &#176;C in the hybridization station.</li>
		<li>Remove the coverslip and wash the slides for 5 min in DPBS at RT. Fixate the sample for 5 min with 1% formaldehyde at RT. At this stage, the material is not yet fully attached to the glass slides and should therefore not be placed on a shaking platform.</li>
		<li>Wash the slides for 5 min in DPBS at RT, followed by dehydration in subsequent 70%, 80%, 90%, and 100% ethanol for 2 min each. Air-dry the dehydrated sample and hybridize it with fluorescently labeled probes.</li>
		<li>Select a centromere-specific probe for chromosome X and another chromosome-specific centromeric probe as a control to determine the X chromosomal content of ovarian cells. In this case, centromere-specific probes for chromosome X (CEP X [DXZ1]) directly labeled with fluorochrome SpectrumGreen and chromosome 18 (CEP 18 [D18Z1]) directly labeled with fluorochrome SpectrumOrange are used.</li>
		<li>Add 1 &#181;L of CEP X, 1 &#181;L of CEP 18, and 18 &#181;L of the hybridization buffer to the sample and seal with a coverslip that is glued to the slides to prevent evaporation of the probe during hybridization. Transfer the slides to the hybridization station for denaturation at 73 &#176;C for 3 min, followed by hybridization during an overnight incubation at 37 &#176;C.</li>
		<li>Remove the coverslip and any remaining glue from the slide after hybridization. Wash the slides in 0.4x SSC/0.3% Tween-20 at 72 &#176;C for 2 min, followed by incubation for 1 min in 2x SSC/0.1% Tween-20 at RT.</li>
		<li>Dehydrate the slides by subsequent 2 min incubations in 70%, 80%, 90%, and 100% ethanol and air-dry in the dark. Cover the slides with a mounting medium containing 4&#8242;,6-diamidino-2-phenylindole (DAPI). Keep at -20 &#176;C for at least 10 min before analysis by fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Examine the signal(s) for the X chromosome with a fluorescence microscope linked to an image processing software.
		<ol>
			<li>First, select fluorochrome DAPI.
			<ol>
				<li>Acquire an image by selecting New Cell &#62; Live &#62; Capture at 630x magnification. A new window with the threshold will appear. Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.</li>
				<li>A new window with enhancement will appear with a suggestion for darker/brighter (12), radius (3), and depth (1). Use the suggested values or adjust them if not satisfied.</li>
			</ol>
			</li>
			<li>Secondly, select fluorochrome SpectrumOrange and click on Capture.
			<ol>
				<li>Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.</li>
				<li>The window with enhancement will appear with a suggestion for darker/brighter (-11), radius (2.7), and depth (0.6). Use the suggested values or adjust them if not satisfied.</li>
			</ol>
			</li>
			<li>Finally, select fluorochrome SpectrumGreen and click on Capture.
			<ol>
				<li>Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.</li>
				<li>The window with enhancement will appear with a suggestion for darker/brighter (0), radius (3), and depth (0). Use the suggested values or adjust it if not satisfied. Save the image in a newly created file. 
				NOTE: Somatic cells were only evaluated when two signals of the control chromosome 18 were visible. In most oocytes, only one signal could be detected for each chromosome.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Store the slides in the dark at 4 &#176;C after analysis to prevent loss of signals.</li>
	</ol>
	</li>
</ol>
2. FISH on paraffin sections of grafted ovarian cortex tissue
NOTE: One fragment of cryopreserved/thawed ovarian cortex tissue of 18 females with TS was xenografted into severe combined immunodeficient (SCID) mice for 5 months. The procedure of xenografting has been described previously and was conducted at the Universit&#233; Catholique de Louvain (Brussels, Belgium) following the local guidelines of the Committee on Animal Research regarding animal welfare (reference 2014/UCL/MD/007)18,19.
<ol>
	<li>Selecting sections of xenografted ovarian cortex tissue containing follicles
	<ol>
		<li>Fixate xenografted ovarian cortex tissue in 4% formaldehyde and embed the tissue in paraffin. Trim the blocks with a scalpel to remove extra paraffin and cut the paraffin block to 4 &#181;m thickness on a rotation microtome.</li>
		<li>Select every seventh section of the paraffin ribbon for hematoxylin and eosin (HE) staining to determine which sections contain follicles. Put the section in a water bath at 40-45 &#176;C and mount them on immunohistochemistry microscope slides.</li>
	</ol>
	</li>
	<li>Deparaffinization and HE staining
	<ol>
		<li>Put the slides on a stove for 10 min at 60 &#176;C, and thereafter immerse the slides in 100% xylene for 5 min. It is not necessary to place the slides directly on the stove. Hydrate the sections for 15 s in 100% ethanol, followed by 2 x 15 s in 96% ethanol. Rinse the slides in tap water for 2 min.</li>
		<li>Stain the slides in hematoxylin for 10 min and thereafter rinse the slides briefly in tap water. Briefly immerse the slides in a bicarbonate solution (100 g of magnesium sulfate and 10 g of sodium bicarbonate in 5 L of distilled water). Rinse the slides in tap water for 5 min.</li>
		<li>Counterstain the slides with eosin for 4 min and dehydrate the slides three times with 100% ethanol, followed by xylene. Coverslip the HE stains on the slides and evaluate the HE sections under a light microscope to select the sections with follicles (100x magnification).</li>
	</ol>
	</li>
	<li>Pre-treatment and hybridization of paraffin sections for DNA FISH
	<ol>
		<li>Select new sections that lay before or after the section that contained follicles from the paraffin ribbon. Mount one section on a glass slide. Dry the paraffin sections for at least 45 min on a stove at 56 &#176;C. It is not necessary to place the slides directly on the stove.</li>
		<li>Deparaffinize the sections in xylene for 10 min. Immerse the slides in 99.5% ethanol and rinse them for 5 min in tap water. Pre-treat the slides with target retrieval solution (low pH) for 10 min at 96 &#176;C. After cooling down, rinse the slides in distilled water.</li>
		<li>Treat the slides for 5 min with 0.01 M hydrochloric acid, followed by pepsin digestion (200 U/mL) for 15 min at 37 &#176;C. Rinse the slides again in 0.01 M hydrochloric acid and subsequently in PBS.</li>
		<li>Fixate the slides in 1% formaldehyde/PBS for 5 min. Rinse the slides briefly in PBS, and then again in demi water. Dehydrate the slides in 99.5% ethanol and let them air-dry.</li>
		<li>Select a centromere-specific probe for chromosome X and another chromosome-specific centromeric probe as a control to determine the X chromosomal content of granulosa cells. Here, chromosome 18 is used as a control.</li>
		<li>Apply 5 &#181;L of probe CEP 18 (D18Z1) directly labeled with fluorochrome SpectrumGreen and probe CEP X (DXZ1) directly labeled with fluorochrome SpectrumRed on the pre-treated slides. Apply a coverslip and seal the area with photo glue. Place the slides in a hybridizer for denaturation at 80 &#176;C for 10 min and hybridization overnight at 37 &#176;C.</li>
		<li>The next day, rinse the slides for 5 min in 2x SSC at 42 &#176;C, followed by a 2 min and a 1 min rinse in 0.3% Nonidet P40 at 73 &#176;C. Refresh the 2x SSC and rinse the slides again for 5 min at room temperature. Cover the cuvette so that the sections are kept in the dark.</li>
		<li>Rinse the slides briefly in distilled water. Dehydrate the slides in 99.5% ethanol and let them air-dry again. Finally, mount the slides with a solution containing DAPI and mounting medium.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Analyze the results under a fluorescence microscope at 630x magnification. Open the image processing software on the computer. Select FISH as the profile.</li>
		<li>Check if the DAPI emission is set at 431 nm and the excitation at 359 nm, Texas red emission at 613 nm and excitation at 595 nm, and FITC emission at 519 nm and excitation at 495 nm.</li>
		<li>Acquire an image by selecting Live &#62; Capture Single Image. Optimize the image quality by adjusting the exposure and gain by moving the Exposure Slider and Gain Slider in the Image menu on the left (e.g., exposure: 212 ms, and gain: 7.9). The required exposure and gain can vary per image; observe the changes during this process to obtain an optimized image. Save the image in a newly created file.</li>
		<li>Store the slides in the dark at 4 &#176;C after analysis to prevent loss of signals.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

FISH analysis is a well-known technique to detect X chromosomal aberrations in lymphocytes or buccal cells of both males and females10. Several studies have described FISH on gametes of males with X chromosomal aberrations, but detailed information obtained by FISH on ovarian cells from females with X chromosomal aberrations is still lacking14. This article presents novel methods based on FISH to determine if aneuploidy is present in the ovarian cells of non-grafted and gra...

Infertility is a health issue of the male or female reproductive system, affecting approximately 186 million individuals of reproductive age worldwide1. In at least 35% of infertile couples, infertility is caused by a disorder of the female reproductive system2. There are many factors that can cause female infertility, such as genetic factors, genital tract abnormalities, endocrine dysfunction, inflammatory diseases, and iatrogenic treatment3.
Genetic abnormalities are present in approximately 10% of infertile females4,5. Of all genetic abnormalities, X chromosome aberrations are the most common cause of ovarian dysgenesis2. Several studies have reported that X chromosomal aberrations in females with Turner syndrome (TS) or Triple X syndrome are associated with premature ovarian failure due to an accelerated loss of germ cells or impaired oogenesis6,7,8.
Aberrations of the X chromosome can be divided into: 1) numerical aberrations, in which the number of X chromosomes is different but the X chromosomes are intact; and 2) structural aberrations, in which the X chromosome has gained or lost genetic material3,9. Numerical aberrations of the X chromosome are more common than structural abnormalities and are often caused by spontaneous errors during cell division3,9. When such an error occurs during meiosis, it may lead to aneuploid gametes and ultimately to offspring with chromosomal aberrations in all cells. When chromosome defects arise in somatic cells as a result of errors occurring during mitosis in the early stages of ontogenesis, it may lead to mosaicism. In these individuals, both cells with normal X chromosomal content and cells with X chromosomal aberrations are present.
In the 1980s, a cytogenetic technique called fluorescence in situ hybridization (FISH) was developed to visualize and locate specific nucleic acid sequences on metaphase and interphase chromosomes10,11. This technique uses fluorescent-labeled DNA probes to bind to a specific sequence in the chromosome, which can then be visualized by using a fluorescence microscope.
Nowadays, FISH is widely used as a clinical diagnostic tool and is considered the gold standard in detecting chromosomal aberrations10. In the field of reproductive medicine, FISH analysis on sperm has been used to gain insight into the X chromosomal content of spermatozoa in males with numerical or structural chromosomal aberrations in somatic cells12,13,14. These studies showed that males with chromosomal aberrations were more likely to have a higher frequency of aneuploid spermatozoa present in their semen compared to males with normal karyotypes12,13,14.
In contrast to spermatozoa, very little is known about the X chromosomal content of ovarian cells (including oocytes, granulosa/theca cells, and stromal cells) in individuals with a chromosomal aberration, as well as the possible consequences of aneuploidy of these cells on their reproductive potential. An important reason for the scarce information on the karyotype of ovarian cells compared to spermatozoa is the fact that women have to undergo an invasive procedure such as a follicle puncture or surgery to obtain oocytes or ovarian cortex tissue. Female gametes are, therefore, difficult to obtain for research purposes.
Currently, an observational intervention study is being performed in the Netherlands to explore the efficacy of ovarian tissue cryopreservation in young females with TS15. One fragment of the ovarian cortex tissue of the patient was available to identify the X chromosomal content of the ovarian cells16,17. As part of the study, a novel method was developed based on FISH of dissociated ovarian cortex tissue to determine if chromosomal aberrations are present in ovarian cells in females carrying a chromosomal aberration in non-ovarian somatic cells, such as lymphocytes or buccal cells. In addition, the effect of aneuploidy in ovarian cells on folliculogenesis was determined as well. To this end, an established FISH protocol was modified that enables the analysis of histological sections of ovarian cortex tissue after artificially induced folliculogenesis during long term xenotransplantation in immunocompromised mice. In this study, we present two methods based on FISH to determine the X chromosomal content in ovarian cells in non-grafted and grafted ovarian cortex tissue in females with X chromosomal aberrations, with the aim to improve basic science on this topic.

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exploring x chromosomal aberrations in ovarian cells by using fluorescence in situ hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

FISH on isolated ovarian cells prior to grafting 
Cryopreserved ovarian cortex tissue from females with 45,X/46,XX (patient A) or 45,X/46,XX/47,XXX (patient B) TS were used to illustrate the results using this protocol. In patient A, 50% of the lymphocytes had a 45,X karyotype and 50% had 46,XX. In patient B, 38% of the lymphocytes were 45,X, 28% were 46,XX, and 34% were 47,XXX. Centromere-specific probes for chromosome X (green) and chromosome 18 as the control (red) were used to determine the X ch...

Watch this Scientific Journal Video about Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization at JoVE.com

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

exploring-x-chromosomal-aberrations-ovarian-cells-using-fluorescence

Research

JoVE Journal

Genetics

945 Views.  Radboudumc. This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Video: Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...