シロアリ全腸のDNA抽出のための手続き型の要約： <ol><li>氷上で冷やしてシロアリは、滅菌ピンセットを用いて消化管削除し、バッファ内の腸のサンプルを安定化させる。 </li><li> PVPP / SDS /フェノールバッファーでサンプルをホモジナイズする。 </li><li>キアゲンDNeasyカラムを用いて粗ライセートからDNAを抽出し精製する。 </li></ol>プロトコル： <ol><li>氷の上に、滅菌ピンセットを使用してワーカーカーストのシロアリから内臓を取り除く。 </li><li>すぐに50μLの氷冷した1 ×分子生物学グレードのTE緩衝液（1 mMトリス- HCl、0.1mMのEDTA、pH8.0）を含有する滅菌、ヌクレアーゼフリーのチューブに内臓との内容を転送します。 -20℃でサンプルを凍結、または均質化と直接進みます。 </li><li>無菌ジルコニア/シリカビーズ500mgの（0.1 mm）を、1％w / vのポリビニルポリピロリドン（PVPPを含む1 × TEバッファー700μlのが事前にロードされた滅菌、ヌクレアーゼフリーの2 mlのスクリューキャップチューブに腸サンプルとバッファーを転送する）。 </li><li> 20％のドデシル硫酸ナトリウム（SDS）を50μlとサンプルへのフェノールの500μlを添加します。 </li><li> 30秒の均質化の3サイクルと氷で冷却の30秒を使用して、最高の設定で（ビーズ拍）ホモジナイズする。 </li><li> 8000 x gで1分間堆積物不溶性物質。 </li><li>粗溶解物の精製のために説明した方法を使用してQiagen社DNeasy列を含む水溶液（最）層の300μlをして浄化する。 </li><li>後で使用するまで-20℃で核酸含有量および凍結試料を定量化する。 </li></ol>

<ol>
	<li>Berthelet, M., Whyte, L. G., Greer, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+Direct+Extraction+of+DNA+from+Soils+for+PCR+Analysis+using+Polyvinylpolypyrrolidone+Spin+Columns.">Rapid, Direct Extraction of DNA from Soils for PCR Analysis using Polyvinylpolypyrrolidone Spin Columns.</a> FEMS Microbiol. Lett. 138, 17-22 (1996).</li><li>Kudo, T., Ohkuma, M., Moriya, S., Noda, S., Ohtoko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Phylogenetic+Identification+of+the+Intestinal+Anaerobic+Microbial+Community+in+the+Hindgut+of+the+Termite,+Reticulitermes+Speratus,+without+Cultivation.">Molecular Phylogenetic Identification of the Intestinal Anaerobic Microbial Community in the Hindgut of the Termite, Reticulitermes Speratus, without Cultivation.</a> Extremophiles. 2, 155-161 (1998).</li><li>Mor&#233;, M. I., Herrick, J. B., Silva, M. C., Ghiorse, W. C., Madsen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Cell+Lysis+of+Indigenous+Microorganisms+and+Rapid+Extraction+of+Microbial+DNA+from+Sediment.">Quantitative Cell Lysis of Indigenous Microorganisms and Rapid Extraction of Microbial DNA from Sediment.</a> Appl. Environ. Microbiol. 60, 1572-1580 (1994).</li><li>Odelson, D. A., Breznak, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+Fatty+Acid+Production+by+the+Hindgut+Microbiota+of+Xylophagous+Termites.">Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites.</a> Appl. Environ. Microbiol. 45, 1602-1613 (1983).</li><li>Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Digital+PCR+Enables+Multigene+Analysis+of+individual+Environmental+Bacteria.">Microfluidic Digital PCR Enables Multigene Analysis of individual Environmental Bacteria.</a> Science. 314, 1464-1467 (2006).</li><li>Purdy, K. J., Embley, T. M., Takii, S., Newdell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Extraction+of+DNA+and+rRNA+from+Sediments+by+a+Novel+Hydroxyapatite+Spin-Column+Method.">Rapid Extraction of DNA and rRNA from Sediments by a Novel Hydroxyapatite Spin-Column Method.</a> Appl. Environ. Microbiol. 62, 3905-3907 (1996).</li><li>Salmassi, T. M., Leadbetter, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Genes+of+Tetrahydrofolate-Dependent+Metabolism+from+Cultivated+Spirochaetes+and+the+Gut+Community+of+the+Termite+Zootermopsis+Angusticollis.">Analysis of Genes of Tetrahydrofolate-Dependent Metabolism from Cultivated Spirochaetes and the Gut Community of the Termite Zootermopsis Angusticollis.</a> Microbiology. 149, 2529-2537 (2003).</li><li>Schmitt-Wagner, D., Friedrich, M. W., Wagner, B., Brune, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+Diversity,+Abundance,+and+Axial+Distribution+of+Bacteria+in+the+Intestinal+Tract+of+Two+Soil-Feeding+Termites+(Cubitermes+spp).">Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp).</a> Appl. Environ. Microbiol. 69, 6007-6017 (2003).</li><li>Tsai, Y. L., Olson, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Method+for+Separation+of+Bacterial+DNA+from+Humic+Substances+in+Sediments+for+Polymerase+Chain+Reaction.">Rapid Method for Separation of Bacterial DNA from Humic Substances in Sediments for Polymerase Chain Reaction.</a> Appl. Environ. Microbiol. 58, 2292-2295 (1992).</li></ol>

我々の経験では、Zootermopisのnevadensisのような木製の餌のシロアリの種の微生物群集から抽出したDNAは、抽出と精製1ラウンド後のPCRテンプレート用に十分に純粋である。しかし、このようなゴミ給と土壌栄養の種など、一部のシロアリはその腸の内容フミン酸の高い濃度を有することができるし、腸内微生物のDNAの追加の精製が必要な場合があります。 5 Z.のnevadensis労働者の内臓からの全DNAの収量は10〜30μg?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PVPP/SDS/phenol</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>homogeneization buffer</td>
</tr>
<tr>
<td>DNeasy Tissue Kit</td>
<td>Kit</td>
<td>Qiagen</td>
<td>77607</td>
<td>Used according to the protocol for isolation of genomic DNA from crude lysates (Appendix H, product manual version: July 2003)</td>
</tr>
<tr>
<td>TE</td>
<td>Buffer</td>
<td>Sigma</td>
<td>T9285</td>
<td>1x buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) from 100x concentrate</td>
</tr>
<tr>
<td>zirconia/silica beads </td>
<td>Supplies</td>
<td>BioSpec Products</td>
<td>11079101z</td>
<td>0.1 mm</td>
</tr>
<tr>
<td>PVPP</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6755</td>
<td>1% w/v polyvinylpolypyrrolidone prepared from dry reagent as a 1x suspension in TE buffer </td>
</tr>
<tr>
<td>Zootermopsis nevadensis</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Termites</td>
</tr>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4390</td>
<td>Sodium dodecyl sulfate 20% soln. in water from dry reagent</td>
</tr>
<tr>
<td>Phenol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>77607</td>
<td>TE-saturated, ~73%</td>
</tr>
<tr>
<td>MiniBeadbeater-8</td>
<td>Tool</td>
<td>BioSpec Products</td>
<td>963</td>
<td>&nbsp;</td>
<tr>
<td>BSS</td>
<td>Buffered Salt Solution, pH 7.2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Formulation per liter: 2.5 g K2HPO4, 1.0 g KH2PO4, 1.6 g KCl, 1.4 g NaCl, 0.075 g CaCl, 1 g MgCl, and 10 mL of a 1M soln. of NaHCO3</td>
</tr>
<tr>
<td>AxioPlan-2</td>
<td>Microscope</td>
<td>Carl Zeiss, Inc. USA</td>
<td>&nbsp;</td>
<td>Outfitted with 40x objective, 1.6x optivar and 10x ocular lenses. Samples were viewed using phase contrast illumination</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

extracting dna from the gut microbes of the termite (zootermopsis angusticollis) and visualizing gut microbes

シロアリは、単独で消費する木材によって内在する能力を持つことが知られているいくつかの動物の一つです。シロアリ腸管は主に酢酸にリグノセルロースの分解を補助する高密度と種が豊富な微生物群集が含まれている、シロアリの代謝を加速させる重要な栄養素（Odelson＆Breznak、1983）。これらの微生物集団内細菌、メタン生成古細菌と、いくつか（&quot;低&quot;）シロアリで、原生動物真核生物です。このように、シロアリは微生物種と彼らはホストの利益のために行う数々の生化学的機能間の相互作用を研究するための優秀な研究テーマです。シロアリの腸だけでなく、様々な生化学的プロセスに関与する重要な遺伝子の微生物集団の種組成は、分子技術を（。シュミット - Wagnerら、2003;。Salmassi＆レッドベター、2003工藤ら、1998）を使用して検討されている。これらの技術は、シロアリ腸内の環境から高品質の核酸の抽出と精製に依存する。 。Bertheletら、1996;。パーディら、1996;。Salmassi＆レッドベター、このビデオで説明されている抽出技術は、環境試料（モルら、1994からの核酸の抽出と精製のために開発されたプロトコルの変更をまとめたものです2003;。オッテセンら、2006）、それはポリメラーゼ連鎖反応（PCR）のテンプレートとして使用するのに適してシロアリの後腸の材料からDNAを生成します。

Watch this Scientific Journal Video about Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes at JoVE.com

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

このビデオでは、シロアリの後腸内に常駐微生物の種からDNAを抽出する手法を示しています。腸内の微生物群集を視覚化するのに便利ですウェットマウントスライドの準備も図示され、そして種が豊富な腸内環境を経由してツアーが与えられます。

シロアリの腸内微生物（Zootermopsis Angusticollis）からDNAを抽出し、腸内微生物の可視化

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

extracting-dna-from-gut-microbes-termite-zootermopsis-angusticollis

Research

JoVE Journal

Biology

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

15.4K ビュー.  California Institute of Technology - Caltech. このビデオでは、シロアリの後腸内に常駐微生物の種からDNAを抽出する手法を示しています。腸内の微生物群集を視覚化するのに便利ですウェットマウントスライドの準備も図示され、そして種が豊富な腸内環境を経由してツアーが与えられます。

ビデオ: Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

インタビュー - シロアリ後腸内微生物群集の調査

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

生物学

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

共生の層 - シロアリ後腸の微生物コミュニティの可視化

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).
In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni, Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.
In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).

原虫シロアリ腸に対するリガンド - 溶解性ペプチドのProtozoacidal活性を試験する in vitroで（原生動物文化）と in vivoで（シロアリ後腸へのマイクロインジェクション）

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

ワイルド毛虫から血液細胞を抽出するための単純なプロトコル

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

ジェネラリストの腸内で代謝的に活性な細菌の同定<em&gt;ハスモンヨトウリットラリス</em&gt; DNAを安定同位体を経由して使用するプロービング<sup&gt; 13</sup&gt; C-グルコース

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

レトロウイルス統合サイトの増幅、次世代シーケンシング、およびゲノムDNAのマッピング

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

マウスの腸内細菌叢に及ぼすバクテリオシンの影響を評価する方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

活性酸素種感受性蛍光発生プローブを用いたマウス腸管オルガノイドの酸化ストレス解析

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

成体 ショウジョウバエ 腸における腸酸性化のモニタリング

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

葉甲虫の宿主 - 腸内微生物叢相互作用研究のための組織培養苗木を用いた軸状昆虫の調製および飼育

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...