<meta charset="utf8"></head><body>用于微生物组分析的高精度蚊子器官分离

<ol>
	<li>Achee, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+strategies+for+mosquito-borne+arbovirus+control.">Alternative strategies for mosquito-borne arbovirus control.</a> PLOS Negl Trop Dis. 13 (1), e0006822 (2019).</li><li>Hoffmann, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+establishment+of+Wolbachia+in+Aedes+populations+to+suppress+dengue+transmission.">Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission.</a> Nature. 476 (7361), 454-457 (2011).</li><li>Porter, J., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+lives+of+Wolbachia.">The cellular lives of Wolbachia.</a> Nat Rev Microbiol. 21 (11), 750-766 (2023).</li><li>Coon, K. L., Brown, M. R., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquitoes+host+communities+of+bacteria+that+are+essential+for+development+but+vary+greatly+between+local+habitats.">Mosquitoes host communities of bacteria that are essential for development but vary greatly between local habitats.</a> Mol Ecol. 25 (22), 5806-5826 (2016).</li><li>Didion, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiome+reduction+prevents+lipid+accumulation+during+early+diapause+in+the+northern+house+mosquito,+Culex+pipiens+pipiens.">Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens.</a> J Insect Physiol. 134, 104295 (2021).</li><li>Gabrieli, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+trilogy:+Microbiota,+immunity+and+pathogens,+and+their+implications+for+the+control+of+disease+transmission.">Mosquito trilogy: Microbiota, immunity and pathogens, and their implications for the control of disease transmission.</a> Front Microbiol. 12, 630438 (2021).</li><li>Garrig&#243;s, M., Garrido, M., Panisse, G., Veiga, J., Mart&#237;nez-de La Puente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+West+Nile+Virus+and+the+microbiota+of+Culex+pipiens+vectors:+A+literature+review.">Interactions between West Nile Virus and the microbiota of Culex pipiens vectors: A literature review.</a> Pathogens. 12 (11), 1287 (2023).</li><li>Zheng, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holobiont+perspectives+on+tripartite+interactions+among+microbiota,+mosquitoes,+and+pathogens.">Holobiont perspectives on tripartite interactions among microbiota, mosquitoes, and pathogens.</a> ISME J. 17 (8), 1143-1152 (2023).</li><li>Flores, G. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+dominance+influences+the+Culex+quinquefasciatus+microbiota.">Wolbachia dominance influences the Culex quinquefasciatus microbiota.</a> Sci Rep. 13 (1), 18980 (2023).</li><li>Hegde, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interkingdom+interactions+shape+the+fungal+microbiome+of+mosquitoes.">Interkingdom interactions shape the fungal microbiome of mosquitoes.</a> Anim Microbiome. 6 (1), 11 (2024).</li><li>Suo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+variation+of+midgut+bacterial+diversity+in+Culex+quinquefasciatus+populations+in+Haikou+city,+Hainan+province,+China.">Seasonal variation of midgut bacterial diversity in Culex quinquefasciatus populations in Haikou city, Hainan province, China.</a> Biology. 11 (8), 1166 (2022).</li><li>Gruber-Vodicka, H. R., Seah, B. K. B., Pruesse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=phyloFlash:+Rapid+small-subunit+rRNA+profiling+and+targeted+assembly+from+metagenomes.">phyloFlash: Rapid small-subunit rRNA profiling and targeted assembly from metagenomes.</a> mSystems. 5 (5), e00920-e00920 (2020).</li><li>Gra&#241;a-Miraglia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spirochetes+isolated+from+arthropods+constitute+a+novel+genus+Entomospira+genus+novum+within+the+order+Spirochaetales.">Spirochetes isolated from arthropods constitute a novel genus Entomospira genus novum within the order Spirochaetales.</a> Sci Rep. 10 (1), 17053 (2020).</li><li>Reveillaud, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wolbachia+mobilome+in+Culex+pipiens+includes+a+putative+plasmid.">The Wolbachia mobilome in Culex pipiens includes a putative plasmid.</a> Nat Commun. 10 (1), 1051 (2019).</li><li>Ghousein, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pWCP+is+a+widely+distributed+and+highly+conserved+Wolbachia+plasmid+in+Culex+pipiens+and+Culex+quinquefasciatus+mosquitoes+worldwide.">pWCP is a widely distributed and highly conserved Wolbachia plasmid in Culex pipiens and Culex quinquefasciatus mosquitoes worldwide.</a> ISME Commun. 3 (1), 40 (2023).</li><li>Juma, E. O., Kim, C. -. H., Dunlap, C., Allan, B. F., Stone, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culex+pipiens+and+Culex+restuans+egg+rafts+harbor+diverse+bacterial+communities+compared+to+their+midgut+tissues.">Culex pipiens and Culex restuans egg rafts harbor diverse bacterial communities compared to their midgut tissues.</a> Parasit Vectors. 13 (1), 532 (2020).</li><li>Lee, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GToTree:+a+user-friendly+workflow+for+phylogenomics.">GToTree: a user-friendly workflow for phylogenomics.</a> Bioinformatics. 35 (20), 4162-4164 (2019).</li><li>Parks, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complete+domain-to-species+taxonomy+for+bacteria+and+Archaea.">A complete domain-to-species taxonomy for bacteria and Archaea.</a> Nat Biotechnology. 38 (9), 1079-1086 (2020).</li></ol>

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			<td>Alcohol 96&#176;</td>
			<td>Fisher scientific</td>
			<td>10332562</td>
			<td></td>
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		<tr>
			<td>Binocular magnifier&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>RAJA</td>
			<td>145517</td>
			<td>150 tablets of 1.5 g</td>
		</tr>
		<tr>
			<td>DNA prep kit</td>
			<td>Illumina</td>
			<td>Provided by MGX sequencing platform</td>
			<td>Previously known as Nextera DNA Flex</td>
		</tr>
		<tr>
			<td>DNA-RNA Shield (50 mL)</td>
			<td>Zymo research</td>
			<td>ZR1100-50</td>
			<td>Preservation buffer</td>
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		<tr>
			<td>DNeasy Blood and Tissue Kit&#160;</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>DNA extraction kit</td>
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			<td>Filter tips 20 &#181;L</td>
			<td>Starlab</td>
			<td>S1120-3810</td>
			<td></td>
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			<td>Filter tips 200 &#181;L</td>
			<td>Starlab</td>
			<td>S1120-8710</td>
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			<td>Filter tips 1000 &#181;L</td>
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			<td>S1122-1730-C</td>
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			<td>Forceps</td>
			<td>FST (Fine Science Tools)</td>
			<td>11252-20</td>
			<td>Dumont Forceps #5</td>
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		<tr>
			<td>Library quantification kit</td>
			<td>Roche</td>
			<td>Provided by MGX sequencing platform</td>
			<td>KAPA Library Quanitification Kits</td>
		</tr>
		<tr>
			<td>Micropipettes 2-20 &#181;L</td>
			<td>Eppendorf</td>
			<td>6.291704</td>
			<td></td>
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		<tr>
			<td>Micropipettes 20-200 &#181;L</td>
			<td>Eppendorf</td>
			<td>6.291703</td>
			<td></td>
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			<td>Micropipettes 100-1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>7.648488</td>
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			<td>Microscope slides&#160;</td>
			<td>Epredia</td>
			<td>J1800BMNZ</td>
			<td>dimension : 75 mm x 50 mm</td>
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		<tr>
			<td>Needles</td>
			<td>Terumo</td>
			<td>AN*2719R1</td>
			<td></td>
		</tr>
		<tr>
			<td>NGS kit&#160;</td>
			<td>Agilent</td>
			<td>Provided by MGX sequencing platform</td>
			<td>Fragment Analyzer Systems HS Genomic DNA 50kb Kit</td>
		</tr>
		<tr>
			<td>NovaSeq 6000</td>
			<td>Illumina</td>
			<td>Provided by MGX sequencing platform</td>
			<td>Sequencer</td>
		</tr>
		<tr>
			<td>PBS Phosphate Buffered Saline (Sterile)</td>
			<td>Fisher scientific</td>
			<td>10212990</td>
			<td></td>
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			<td>Permanent black marker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Eppendorf&#160;</td>
			<td>Dutscher</td>
			<td>33871</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Support for needles</td>
			<td>FST (Fine Science Tools)</td>
			<td>26016-12</td>
			<td>Moria MC1 Pin Holder 12 cm</td>
		</tr>
	</tbody>
</table>

dissection of mosquito ovaries, midgut, and salivary glands for microbiome analyses at the organ level

<meta charset="utf8"></head><body>作者聚焦：优化蚊子器官解剖以研究共生体和媒介微生物组

解剖蚊子卵巢、中肠和唾液腺，用于器官水平的微生物组分析

The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a ...

dissection-mosquito-ovaries-midgut-salivary-glands-for-microbiome

Research

JoVE Journal

Biology

Dissection of Mosquito Ovaries, Midgut, and Salivary Glands for Microbiome Analyses at the Organ Level

1.3K 次观看.  University of Montpellier, INRAe, CNRS, IRD. 我们的研究重点是使用宏基因组分析在单个器官规模上蚊子中的微生物组群落，我们研究卵巢、中肠和唾液腺中的共生体。通过防止解剖过程中的交叉污染，我们的目标是研究这些微生物如何在前沿尺度上影响蚊子生物学。以及添加有针对性的控制方法。处理单个器官真的很复杂，因为我们需要完整地处理它们，而不会在器官之间造成任何损坏...

视频: 作者聚焦：优化蚊子器官解剖以研究共生体和媒介微生物组

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &amp;#955; DNA at the Single Molecule Level

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...