Name: visualization of microbiota in tick guts by whole-mount in situ hybridization
Uploaded: 2018-06-01T14:45:00
Description: Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

We sincerely thank Dr. Mustafa Khokha, Yale University, for providing the use of his laboratory resources. We are grateful to Mr. Ming-Jie Wu for excellent technical assistance. EF is an HHMI investigator.This work was supported by a gift from the John Monsky and Jennifer Weis Monsky Lyme Disease Research Fund.

1. Preparation of DNA Templates
<ol>
	<li>Download the 16S rRNA sequence specific to each bacterial genera from the NCBI database and design polymerase chain reaction (PCR) compatible forward and reverse primers that will amplify genera-specific regions (~200 - 250 base-pair long) as described earlier19. Also refer to the open source databases SILVA20,21 and probeBase22 online resources for rRNA-targeted oligonucl...

<ol>
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This is the first use of a whole-mount in situ hybridization (WMISH) technique to study the microbiota of an arthropod vector of pathogens. Our protocol was adapted from one used to study development in Drosophila and in frog embryos25,26. Whole-mount RNA in situ hybridization has been routinely used to localize gene transcripts spatially and temporally27 and visualization of transcripts can be done by bright-fie...

Human and livestock pathogens transmitted by arthropod vectors are found worldwide and account for about 20% of infectious diseases globally1, but effective and safe vaccines against most of these pathogens are not available. Our understanding of the important role of commensal, symbiotic and pathogenic microorganisms, collectively known as the microbiome2, in modulating and shaping the health of almost all metazoans3 is expanding. It is now evident that arthropod vectors of pathogens also harbor gut microbiota and these vector-associated microbiota have been shown to influence diverse vector-borne pathogens4,5. The arthropod microbiome is composed of eubacteria, archaea, viruses, and eukaryotic microbes such as protozoa, nematodes, and fungi6. However, the predominant research focus has been on eubacteria due, in part, to the availability of marker genes and reference databases to identify specific bacterial members.
With a focus on Ixodes scapularis, the tick vector of multiple human pathogens including Borrelia burgdorferi7, the causative agent of Lyme disease, the optimization of a microbial visualization technique was aimed at improving our understanding of tick gut microbiota in the context of vector-pathogen interactions. Several questions remain to be answered in the tick microbiome field. The gut is the site of the first extended encounter between the tick and the incoming pathogen in the context of horizontally transferred pathogens; therefore, understanding the role of vector gut microbiota in modulating vector-pathogen interactions will reveal meaningful insights. Ticks have a unique mode of blood meal digestion, where processing of blood meal components takes place intracellularly8. The gut lumen seemingly serves as a vessel to contain the blood meal as the tick feeds, and nutrient digestion and assimilation ensue throughout the several days of feeding and continue post-repletion. The pathogens acquired by the tick during feeding enter the gut lumen along with the bloodmeal and thus the lumen becomes a primary site of interactions among the tick, pathogen, and resident microbiota. As digestion proceeds through the repletion and Ixodid tick molting, the gut undergoes structural and functional changes9. The composition and the spatial organization of gut bacteria is also likely to vary in concert with the changing gut milieu. It is, therefore, important to understand the architecture of resident bacteria in the tick gut to fully understand the interplay of tick, pathogen, and gut microbiota.
Molecular techniques to describe host-associated microbiota routinely utilize high-throughput parallel sequencing strategies10 to amplify and sequence bacterial 16S ribosomal DNA (rDNA). These sequencing strategies circumvent the need to obtain axenic cultures of specific bacteria and provide an in-depth description of all bacterial members represented in the sample. Nevertheless, such strategies are confounded by the inability to distinguish environmental contaminations from bona fide residents. Further, when assessing samples, such as ticks, that are small in size and hence contain low microbiota-specific DNA yields, the likelihood of amplification of environmental contaminants is increased11 and results in the ambiguous interpretation of microbiome composition. Functional characterization in conjunction with the visualization of specific viable bacteria will, therefore, be critical to define and discern the microbiome of the tick temporally and spatially. Towards this goal, we took advantage of the whole-mount RNA in situ hybridization. This technique is routinely used to assess gene expression patterns in organs and embryos12,13,14 and allows semiquantitative analysis of expression over the entire sample of interest. This differs from traditional in situ hybridization techniques which utilize tissue sections and often require extensive analysis of sectioned material with a computational assembly to predict expression in whole organs15. While whole-mount generally refers to whole organisms12, here whole-mount refers to whole guts or organs. The advantages of using the whole-mount RNA in situ hybridization approach to assess the architecture of tick gut microbiota are multifold. The tick gut is composed of 7 pairs of diverticula, each pair varying in size16. The functional differences, if any, among these diverticula, are not understood in the context of tick biology, tick microbiota or tick-pathogen interactions. Manipulations of the gut that rupture the gut diverticula would displace microbiota present in the gut lumen or those associated loosely with the gut and result in misinterpretation of the spatial localization of microbiota. Fluorescence-labeled RNA in situ hybridization has been utilized earlier to examine tick gut transcripts17 by fixing and opening individual gut diverticula to ensure probe hybridization and to localize B. burgdorferi transcripts by sectioning paraffin-embedded whole ticks18. Both these approaches require manipulations of the tick tissues prior to hybridization that would affect the gut microbiota architecture.
In this report, we describe in detail the protocol to examine viable tick gut microbiota using whole-mount in situ hybridization (WMISH). The use of whole-mount RNA in situ hybridization enables a global understanding of the presence and abundance of specific gut bacteria in the different regions of the gut and may spur new insights into tick gut biology in the context of pathogen colonization and transmission. Further, the use of RNA probes directed against specific bacterial RNA allows detection of viable bacteria in the tick gut.

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			<td height="21" style="height:21px;width:320px;">pGEM-T Easy Vector System</td>
			<td style="width:220px;">Promega</td>
			<td style="width:348px;">A1360</td>
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			<td height="21" style="height:21px;width:320px;">Digoxygenin-11-UTP</td>
			<td style="width:220px;">Roche</td>
			<td align="right" style="width:348px;">1209256910</td>
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			<td height="21" style="height:21px;width:320px;">dNTP</td>
			<td style="width:220px;">New England Biolabs&#160;</td>
			<td style="width:348px;">N0447S</td>
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			<td height="21" style="height:21px;width:320px;">DNAse I(RNAse-free)</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe SP6 RNA synthesis kit</td>
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			<td height="21" style="height:21px;width:320px;">HiScribe T7 High Yield RNA Synthesis Kit</td>
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			<td height="21" style="height:21px;width:320px;">Water, RNase-free, DEPC-treated</td>
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			<td style="width:348px;">AB02128-00500</td>
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			<td height="21" style="height:21px;width:320px;">EDTA, 0.5M, pH 8.0</td>
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			<td height="21" style="height:21px;width:320px;">Formaldehyde, 37%</td>
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			<td height="21" style="height:21px;width:320px;">Formamide</td>
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			<td height="21" style="height:21px;width:320px;">EGTA</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">E-4378</td>
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			<td height="21" style="height:21px;width:320px;">RNA from torula yeast</td>
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			<td height="21" style="height:21px;width:320px;">Heparin, sodium salt</td>
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			<td height="21" style="height:21px;width:320px;">Denhardt&#39;s Solution, 50X</td>
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			<td height="21" style="height:21px;width:320px;">RNase A</td>
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			<td height="21" style="height:21px;width:320px;">RNase T1</td>
			<td style="width:220px;">ThermoScientific&#160;</td>
			<td style="width:348px;">EN0541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Maleic acid</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">M0375-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Blocking reagent</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11096176001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-Digoxigenin-AP, Fab fragments</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Levamisol hydrochloride</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">31742-250MG</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:320px;">Chromogenic substrate for alkaline phosphatase</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11442074001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bouin&#39;s solution</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">HT10132-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hydrogen peroxide</td>
			<td style="width:220px;">Mallinkrodt Baker, Inc</td>
			<td style="width:348px;">2186-01</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Single stranded RNA ladder</td>
			<td style="width:220px;">Ambion -Millenium</td>
			<td style="width:348px;">AM7151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;#11 High-Carbon steel blades&#160;</td>
			<td style="width:220px;">C and A Scientific Premiere</td>
			<td style="width:348px;">#11-9411</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Thermocycler</td>
			<td style="width:220px;">BioRad, CA</td>
			<td align="right" style="width:348px;">1851148</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Spectrophotometer</td>
			<td style="width:220px;">ThermoScientific&#160;</td>
			<td style="width:348px;">NanoDrop 2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Orbital shaker</td>
			<td style="width:220px;">VWR</td>
			<td style="width:348px;">DS-500E Digital Orbital shaker</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Shaking water bath</td>
			<td style="width:220px;">BELLCO Glass, Inc</td>
			<td style="width:348px;">Hot Shaker-7746-12110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Gel&#160; documentation system</td>
			<td style="width:220px;">BioRad</td>
			<td style="width:348px;">Gel Doc XR+ Gel documentation system</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Nikon</td>
			<td style="width:348px;">NikonSM2745T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">AXIO Scope.A1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dissection microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">STEMI 2000-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Light box</td>
			<td style="width:220px;">VWR</td>
			<td style="width:348px;">102097-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">PCR purification kit&#160;</td>
			<td style="width:220px;">Qiagen</td>
			<td align="right" style="width:348px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image capture software</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">Zen lite</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image editing software&#160;</td>
			<td style="width:220px;">Adobe&#160;</td>
			<td style="width:348px;">Adobe Photoshop CS4 version 11.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image analysis software</td>
			<td style="width:220px;">National Institutes of Health</td>
			<td>ImageJ-NIH /imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;">Automation compatible instrumentation</td>
			<td style="width:220px;">Intavis Bioanalytical Instruments, Tubingen, Germany).&#160;</td>
			<td style="width:348px;">Intavis, Biolane HT1.16v</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The measurement and estimation of the quality of the RNA probes are critical prior to beginning the staining. In vitro transcription efficiency depends highly on the amount and quality of the DNA template. We routinely visualized the RNA probes on a formaldehyde gel to verify the purity and amount of probe generated by the transcription reactions. The probes should appear as bright, discrete bands (Figure 2). Spectrophotometric measurements of RNA co...

Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

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.

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 ...

This method can help to answer key questions about tick entomology, such as assessing the presence of viable bacteria in the tick spatially and temporally. The main advantages of this technique are that it does not require sectioning of the ticks or expensive equipments, and that it allows spacial and temporal visualization of gene transcripts. Generally, individuals new to this technique will struggle because it involves multiple steps and reagents.However, this technique is not laborious. Although it can take about three days to complete, hands on time is only a few hours each day. In preparation, feed the collected ticks for 48 to 72 hours before the gut collection.To dissect the gut from the tick add 20 to 30 microliters of MEMFA solution to a clean glass slide. Then place the tick into the MEMFA, and adjust focus to the tick's head. Now, using a pair of sterile high carbon steel razor blades slice off the head region at the scutum leaving the body of the tick in tact.Next, gently press the body to push the gut diverticula and salivary glands out of the body cuticle. Then without damaging the guts, carefully separate them from the salivary glands and carefully scoop the cuts onto a razor blade. Collect all of the harvested guts in a 1.5 milliliter tube in 500 micrometers of MEMFA.Then incubate the tube at room temperature with gentle rocking for one hour. The next day dehydrate the tissue as indicated in the text protocol and store it at minus 20 degrees Celsius. To prepare a mesh basket, first, use a heated single edge blade on a scalpel to cut the bottoms off of a two milliliter or five milliliter snap top centrifuge tube.The blade may need to be reheated several times before the cut is completed. Next, cut a square of a 110 micron nylon mesh that is slightly larger than the new openings. Then, bond the mesh to the opening by lightly pressing the tube into the mesh on a hotplate set to medium/low heat until a good seel is made.After it cools, trim off the excess mesh, and make sure there are not gaps. Make 24 of these baskets to match a 24 well plate. Next, cut holes into the cover of a 24 well plate, such that the lip of the sample basket will grip the hole and not fall through.When completed, the assembled sample baskets should sit all the way into the wells. Now, use this fitted basket holder to transfer baskets from one plate to another with relative ease all through the in situ hybridization. Use two such plates so that while the sample is incubating in one, the other can get prepared for the next wash.This section covers certain steps of the in situ hybridization. See the text protocol for a complete description. To begin, transfer each fixed tissue sample to a labeled sample basket.Organize the plate by experimental conditions, and stain at least five guts per condition. First, gently rehydrate the samples. Avoid introducing air bubbles by gradually increasing the ratio of PBS-T to ethanol in each wash.During the first day, after the two hour pre-hybridization step, instead of discarding the hybridization buffer, store it for future use during the assay. Later, when treating the samples with probe hybridization solution, cover the plate snugly with aluminum foil to prevent evaporation of the solution. On the second day, load the reserved hybridization buffer into the 24 well plate, and warm the plate up to 60 degrees Celsius.Later that day, to remove all traces of probe and hybridization buffer, wash the samples twice with 2x SSC for three minutes per wash. Then wash the samples three times with 2x SSC for 20 minutes per wash at 60 degrees Celsius. Then prepare the primary antibody in blocking buffer.Load 500 microliters of this mixture into each well. After the blocking step, incubate at room temperature for about four hours or at four degrees Celsius overnight. On the third day, run the color reaction in chromogenic substrate.Cover the sample plate with aluminum foil. And during the reaction, periodically check for over staining with the dissection microscope. On the fourth day it is important to incubate and bleach on a light box as this allows for any pigmentation in the samples or coloration from Bouin's solution to be removed, and enhances the probe signal for clear visualization.Then after washing with SSC, carefully transfer the gut samples by inverting the basket into a new 24 well plate. While in wells, thoroughly rinse off the baskets using 70%ethanol. Then precede with imaging the samples.It is critical to estimate the quality of the RNA probes before using them. The probes can be checked on a formaldehyde gel. If they appear as bright, discrete bands they are of good quality.When staining some variation in distribution is normal among biological replicates as well as among the seven pairs of diverticula of individual guts. Therefore, it is best to stain at least five guts per condition to get an idea of average staining pattern. Overall success is best gaged by comparing the staining of sense and antisense samples for each condition.Both must be developed for the same amount of time. The sense samples should have minimal to no purple color, while the antisense samples should display robust staining with minimal background. Critically, the tissues must be fully emersed in the stain for an even exposure.It is important to avoid over development of the color reaction. This can result in a high amount of background staining, and the sense samples may begin to look like antisense samples. Another factor that affects results is how much the ticks are fed beforehand.Ticks that are unfed, or fed for only 24 hours, are difficult to work with. Their guts are more easily damaged and do not stain well. Once mastered, this technique can be done in two and half to three days if it is performed properly.While attempting this procedure, it is important to remember to keep all of the supplies listed in the table of reagents and materials ready before beginning your experiment. Thanks for watching, and good luck with your experiments.

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Research

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Genetics

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 ...

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 ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

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 ...

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 ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

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

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

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

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