We thank Mr. Taku Shimada, the delegate of AntRoom, Tokyo, Japan, for providing us with his photograph of V. emeryi reproductives. This project was funded by the Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists (16J00011), and Grant in Aid for Young Scientists (B)(16K18626).

1. Field Collection and Maintenance of V. Emeryi Colonies in the Laboratory
NOTE: Nests of V. emeryi are found in rotting logs and fallen decaying tree branchesin secondary forests throughout Japan. This species shows two types of colonies, i.e., (1) colonies producing only long-winged queens and (2) colonies mainly producing short-winged queens in addition to small number of long-winged queens8,14. In this protocol, we collected the latter type of colonies in Ishikawa prefecture, Japan.
<ol>
	<li>Collect V. emeryi colonies during early summer. 
	NOTE: To obtain sufficient numbers of sexual individuals during the reproductive season, colonies containing more than 300 individuals are preferred.</li>
	<li>Transfer the ant specimens from the collected branches to an artificial plaster nest with a glass cover using an aspirator (Figure 2, left).</li>
	<li>Maintain colonies in the artificial nest at 25 &#176;C under a 16:8 h light/dark cycle. Provide tap water with a wash bottle to wet the plaster.
	<ol>
		<li>Add about 100 mg of dry cricket powder wrapped in aluminum foil and a brown sugar water-filled tip (20 &#181;L tip) every other day until new reproductives (F1 winged-queens and F1 males) emerge.</li>
	</ol>
	</li>
</ol>
2. Experimental Laboratory Crosses
NOTE: New reproductives start to emerge from late summer to autumn (Figure 3). Long-winged queens are produced sexually, and short-winged queens are produced clonally and have the maternal genome (Figure 1). Use long-winged queens and males for inbreeding crosses.
<ol>
	<li>To stop individuals from moving, place colonies in a constant environment room at 4 &#176;C for 15 min.</li>
	<li>Remove the mid-legs of 30 workers using forceps under a stereoscopic microscope and transfer them into new smaller plaster nest (Figure 2, right) for inbreeding crosses. 
	NOTE: The legs are removed to distinguish the present workers from the workers that will be produced by the subsequent inbreeding crosses.</li>
	<li>Add 3-4 larvae or pupae into a plaster nest containing workers. 
	NOTE: Workers show exploratory activity in the F0 queens-less colony. Larvae or pupae can effectively attract these workers and new reproductives in the center of the colony during crossing test. As a result, keep conditions of the experimental colony close to the normal colony.</li>
	<li>Transfer a long-winged queen and a male into a plaster nest prepared in step 2.3 for inbreeding crosses.</li>
	<li>Keep colonies at 25 &#176;C under a 16:8 h light/dark cycle with food and water provided as described in 1.3 until the queen lose her wings and lay eggs. 
	NOTE: This takes one week to a month.</li>
	<li>Check the experimental colony everyday under a stereoscopic microscope. After performing inbreeding crosses between the F1 offspring, eggs can be observed under a stereoscopic microscope.</li>
	<li>After the F1 queen starts laying eggs, remove F1 males and larvae or pupae added in step 2.3 from the nest to avoid mixing of the F1 generation (males and females used for inbreeding crosses) and the F2 generation (offspring produced from inbreeding crosses). 
	NOTE: If there are few males in the colony, it is possible to induce inbreeding crosses using one male and 1 to 3 queens in the same experimental colony.</li>
	<li>Keep colonies under the same conditionsas described in 1.3, until F2 offspring emerge. 
	NOTE: Transfer F1 queen and F2 offspring into new larger plaster nest (Figure 2, left) for long-term colony keeping.</li>
</ol>
3. Evaluation of Inbreeding Success
<ol>
	<li>DNA extraction and genotyping of the parental generation (F0)
	<ol>
		<li>Remove one leg of a F0 queen using forceps and transfer the leg to a 1.5 mL microtube containing 100 &#181;L of chelation agent.</li>
		<li>Under a stereoscopic microscope, dissect a female abdomen in glass dish filled with 300 &#181;L of ultrapure water using forceps and isolate the spermatheca containing the sperm from mated males.</li>
		<li>Peel away the tissue of the spermatheca and isolate the sperm from the tissue of the female using insect pins. 
		NOTE: To facilitate sperm extraction from the spermatheca, store female specimens in 100% EtOH for more than one day before dissection.</li>
		<li>Using a micropipette, transfer the sperm into a 1.5 mL microtube containing 100 &#181;L of chelation agent.</li>
		<li>Incubate samples of F0 queen and sperms prepared in step 3.1.1 and 3.1.3, respectively, at 95 &#176;C for 20 min. Flash centrifuge the microtube and store at 4 &#176;C.</li>
		<li>Genotype all samples using method described elsewhere4.</li>
	</ol>
	</li>
	<li>DNA extraction from the pair of ants used for inbreeding crosses (F1)
	<ol>
		<li>After confirming egg production by the sib mated F1 queen, extract the DNA of the queen using her shed wings or one mid-leg and genotype using same method described in section 3.1 above.</li>
		<li>Extract DNA of F1 male using one leg and genotype them using same method described in section 3.1 above. 
		NOTE: Samples can be stored in 100% EtOH before DNA extraction, and DNA in chelation agent can be stored for two months at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Evaluation of male fertility in males produced from inbreeding crosses 
	NOTE: Diploid males produced from inbreeding crosses are often sterile.
	<ol>
		<li>Dissect internal reproductive organs in a glass dish with 400 &#181;L of PBS solution using forceps.</li>
		<li>Remove PBS and add 4% paraformaldehyde (PFA) using a micropipette.</li>
		<li>Fix the tissue by incubating in PFA for 30 min at room temperature (15 - 25 &#176;C).</li>
		<li>Wash tissue 5 times with 400 &#181;L of PBS using a micropipette.</li>
		<li>Dilute the 4&#39;,6-diamidino-2-phenylindole (DAPI) solution to 1 &#181;g/mL in PBS.</li>
		<li>Remove PBS and add approximately 300 &#181;L of this dilute DAPI staining solution to tissue.</li>
		<li>Incubate 15 min under dark condition at room temperature (15 - 25 &#176;C).</li>
		<li>Wash tissue 5 times with 400 &#181;L of PBS, and transfer tissue on center of slide glass using forceps.</li>
		<li>Mount tissue on mounting medium containing Tetramethylrhodamine (TRITC)-conjugated phalloidin.</li>
		<li>Observe samples by confocal laser scanning microscope using 20X or 63X objective lenses.</li>
		<li>Use a 405 nm excitation laser and a hybrid detector at 410-530 nm for DAPI detection.</li>
		<li>Use a 561 nm excitation laser and a hybrid detector at 565-650 nm for TRITC detection.</li>
		<li>Use a scan speed of 400 Hz (400 lines/s) at a resolution at 1024 &#215; 1024 pixels.</li>
		<li>Capture images using a software platform.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This article demonstrates protocols that can be used to induce inbreeding crosses and evaluate the occurrence of inbreeding in the ant V. emeryi. In the experiments, genotyping of the individuals used for crosses is necessary to ensure that inbreeding crosses were successful. However, the effectiveness of these crossing tests is clearly apparent as diploid males can be produced throughout the year, while haploid males can only be produced in autumn in both the field and the laboratory6. S...

Eusocial Hymenopteran taxa, such as ants and bees, have evolved a haplodiploid sex-determination system in which individuals that are heterozygous at one or more complementary sex determination (CSD) loci become females, while those that are homo- or hemizygous become males (Figure 1A)1.
Genetic and molecular components involved in the sex determination cascade have been well studied in the honeybee, Apis mellifera, a hymenopteran model organism2,3,4. Recent comparative genomics investigations suggest that ants and honeybees share many putative homologs in the sex determination pathway, such as the initial sex determination gene, csd5. However, evidence for the functional conservation of these homologs is still lacking in ants.
To address this problem, inbreeding lines need to be developed as they are essential for genetic mapping and molecular studies. However, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory because of the complex nature of the life cycles that have evolved.
Here, we use Vollenhovia emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ants6,7. The inbreeding lines of this species were developed previously for linkage mapping of quantitative trait loci (QTL) for traits related to sex determination for the first time in ants6. In addition, the molecular sex-determination cascade has been investigated7. This species has evolved an unusual reproduction system that employs both gynogenesis and androgenesis (Figure 1B)8,9. Most new queens and males are clonally produced from the maternal and paternal genomes, respectively. In addition, workers and some queens are produced sexually8. This reproduction system is particularly well suited to genetic studies because the inbreeding crosses produced using sexually produced queens and males are genetically equivalent to a classic backcross. Since sexually produced queens differ morphologically from queens produced from maternal genomes10 (Figure 1B), conducting and evaluating inbreeding crosses is greatly simplified using this method.
In this article, the methods for establishment of laboratory colonies for crossing test, application of inbreeding crosses using full-sib pairs, and evaluating the success of those crosses using genotyping of colony members and dissection of male offspring genitalia are described in V. emeryi.
Regardless of the reproduction system employed, application of inbreeding crosses is often the essential first step in any investigation of sex determination systems in the Hymenoptera. For example in Cardiocondyla obscurior, the almost complete absence of diploid males after 10 generations of full-sib mating in the laboratory demonstrates absence of CSD locus11. It is possible to predict the number of CSD loci from the ratio of males produced in inbreeding crosses6,12,13.

<table border="0" cellpadding="0" cellspacing="0" style="width:1039px;" width="1038">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Plaster powder</td>
			<td style="width:239px;">N/A</td>
			<td style="width:185px;">N/A</td>
			<td style="width:223px;">Any brand can be used</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Charcoal, Activated, Powder</td>
			<td style="width:239px;">Wako</td>
			<td style="width:185px;">033-02117,037-02115</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Slide glass</td>
			<td style="width:239px;">N/A</td>
			<td style="width:185px;">N/A</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Dry Cricket diet</td>
			<td style="width:239px;">N/A</td>
			<td style="width:185px;">N/A</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Brown shuger&#160;</td>
			<td style="width:239px;">N/A</td>
			<td style="width:185px;">N/A</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Styrene Square-Shaped Case</td>
			<td style="width:239px;">AS ONE</td>
			<td style="width:185px;">Any size</td>
			<td>Size varies by number of ants</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Incbator</td>
			<td style="width:239px;"></td>
			<td style="width:185px;"></td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Aluminum block bath Dry thermo unit DTU-1B</td>
			<td style="width:239px;">TAITEC</td>
			<td>0014035-000</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">1.5mL Hyper Microtube,Clear, Round bottom</td>
			<td style="width:239px;">WATSON</td>
			<td>131-715CS</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Ethanol (99.5)</td>
			<td style="width:239px;">Wako</td>
			<td style="width:185px;">054-07225</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Stereoscopic microscope</td>
			<td style="width:239px;">N/A</td>
			<td style="width:185px;">N/A</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Forseps</td>
			<td>DUMONT</td>
			<td style="width:185px;">0108-5-PO</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Chelex 100 sodium form</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:185px;">11139-85-8</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Phosphate Buffer Saline (PBS) Tablets, pH7.4</td>
			<td style="width:239px;">TaKaRa</td>
			<td style="width:185px;">T9181</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">Paraformaldehyde</td>
			<td style="width:239px;">Wako</td>
			<td style="width:185px;">162-16065</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:392px;">-Cellstain- DAPI solution</td>
			<td style="width:239px;">Dojindo Molecular Technologies</td>
			<td>D523</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:392px;">VECTASHIELD Hard&#12539;Set Mounting Medium with TRITC-Phalloidin</td>
			<td style="width:239px;">Vector Laboratories</td>
			<td style="width:185px;">H-1600</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">ABI 3100xl Genetic Analyzer</td>
			<td style="width:239px;">Applied Biosystems</td>
			<td style="width:185px;"></td>
			<td style="width:223px;">Directly contact the constructor formore informations.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:392px;">Confocal laser scanning microscope Leica TCS SP8</td>
			<td style="width:239px;">Leica</td>
			<td style="width:185px;"></td>
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induction and evaluation of inbreeding crosses using the ant, vollenhovia emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Results of microsatellite analysis using F0 and F1 generations showed that inbreeding crosses were produced successfully (Figure 4)6. As a result of inbreeding crosses, mated queens were obtained within one month of establishing the experimental crossing colonies. A quarter (27.1 &#177; 8.91% SD) of all offspring (F2) from the inbreeding crosses was male, while the remainder was female (workers and a qu...

Watch this Scientific Journal Video about Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi at JoVE.com

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

induction-evaluation-inbreeding-crosses-using-ant-vollenhovia

Research

JoVE Journal

Genetics

7.5K Views.  Utsunomiya University. In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

Video: Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

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 ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...