We appreciate the colleagues in Taiwan Cetacean Society, Marine Biology and Cetacean Research Center of National Cheng Kung Univerisy, Farglory Ocean Park, and Animal Disease Diagnostic Center of National Chiayi Universiy for sample collection. This project was funded by grant to WCY from the Council of Agriculture of Taiwan (100AS-02.1-FB99).

<ol>
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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=DNA+surveillance:+web-based+molecular+identification+of+whales,+dolphins,+and+porpoises.">DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.</a> J.Hered. 94, 111-114 (2003).</li><li>Ngom, B., Guo, Y., Wang, X., Bi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+lateral+flow+test+strip+technology+for+detection+of+infectious+agents+and+chemical+contaminants:+a+review.">Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review.</a> Anal. Bioanal. Chem. 397, 1113-1135 (2010).</li><li>Muldoon, M. T., Onisk, D. V., Brown, M. C., Stave, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets+and+methods+for+the+detection+of+processed+animal+proteins+in+animal+feedstuffs.">Targets and methods for the detection of processed animal proteins in animal feedstuffs.</a> Int. J. Food Sci. Technol. 39, 851-861 (2004).</li><li>Rao, Q., Hsieh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+commercial+lateral+flow+feed+test+for+rapid+detection+of+beef+and+sheep+content+in+raw+and+cooked+meats.">Evaluation of a commercial lateral flow feed test for rapid detection of beef and sheep content in raw and cooked meats.</a> Meat Sci. 76, 489-494 (2007).</li><li>Tamura, K., 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=MEGA5:+molecular+evolutionary+genetics+analysis+using+maximum+likelihood,+evolutionary+distance,+and+maximum+parsimony+methods.">MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.</a> Mol. Biol. Evol. 28, 2731-2739 (2011).</li><li>Atassi, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+structure+of+myoglobin:+the+complete+immunochemical+anatomy+of+a+protein+and+conclusions+relating+to+antigenic+structures+of+proteins.">Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins.</a> Immunochemistry. 12, 423-438 (1975).</li><li>Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridoma+technology+for+the+generation+of+monoclonal+antibodies.">Hybridoma technology for the generation of monoclonal antibodies.</a> Methods Mol. Biol. 901, 117-135 (2012).</li><li>Kao, D. J., Hodges, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+a+synthetic+peptide+immunogen+over+a+protein+immunogen+in+the+development+of+an+anti-pilus+vaccine+for+Pseudomonas+aeruginosa.">Advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for Pseudomonas aeruginosa.</a> Chem. Biol. Drug Des. 74, 33-42 (2009).</li><li>Dolar, M. L., Suarez, P., Ponganis, P. J., Kooyman, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+in+pelagic+small+cetaceans.">Myoglobin in pelagic small cetaceans.</a> J. Exp. Biol. 202, 227-236 (1999).</li><li>Lo, C., 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=Rapid+immune+colloidal+gold+strip+for+cetacean+meat+restraining+illegal+trade+and+consumption:+implications+for+conservation+and+public+health.">Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health.</a> PLoS ONE. 8, e60704 (2013).</li></ol>

The authors declare that they have no competing financial interests.

担体タンパク質にコンジュゲートした合成ペプチドを使用することにより、その同族タンパク質と比較して著しくより効果的です。 mAbは既知の相対位置を有するエピトープを使用して開発されているので、サンドイッチに基づく技術については、本研究では2 mAbは、標的抗原エピトープとの互いの相互作用を妨害する可能性はありません。また、天然のタンパク質との反応性及び合成ペプチドコンジュゲートで免疫したマウスの抗体は、天然タンパク質19から製造天然タンパク質と抗体との反応性よりも強いかもしれません。合成ペプチド複合体の使用は、したがって、効果的な免疫手順や適切な抗ペプチドモノクローナル抗体の生成のために推奨されます。 タンパク質の構造は、主にポリペプチド鎖中のアミノ酸の配列を含みます。各アミノ酸は、特定の特性をもたらす、その側鎖を有しており、わずかにアミノACIを変更しますD配列は、構造変化をもたらします。 10〜20アミノ酸長を有するペプチドは、抗体調製のための理想的であるので、C末端領域における合成ペプチド（免疫原）の長さは、コア抗原性領域が認識されることを確実にするために増加しました。したがって、異なるエピトープ構造をもたらす様々な動物のMbpsの間のアミノ酸残基を効率的に区別することができます。例えば、 図3（a）は、上記のペプチド設計は、シール付きマイナスの鯨類が、と強く反応しCGF5H9に貢献示しています。別の例としては、ニワトリ、コア抗原部位2の犬のものと同一の配列を有しているがこれは外側の領域中の配列の違いは構造変化につながるため、可変なかったことを示しCGF5H9の鶏や犬に向かって明確な親和性であります抗原と抗体との間の結合親和性。 ウエスタンブロット、ドットブロット、および間接ELISAはで使用されていました適切なモノクローナル抗体をスクリーニングするための手法。ウェスタンブロットは、広く、組織抽出物またはホモジネート中の特定のタンパク質を検出するために使用されます。この技術では、ゲル電気泳動は、ポリペプチドの長さによって変性タンパク質を分離するために使用されます。したがって、信号は、予測されたタンパク質の分子量を示しているかどうかを確認することが可能です。変性タンパク質を使用するためしかし、検出結果（正または負の、強いまたは弱い信号）は、抗原 - 抗体結合の実際の状況を表していない可能性があります。その結果、ドットブロットは二段目のスクリーニングのために使用することができます。ドットブロットは、タンパク質を検出するための技術です。これは、ウェスタンブロット法の単純化を表します。ドットブロット法では、検出される分子を含む混合物は、ドットなどの膜上に直接塗布されます。タンパク質試料は変性されていないので、これはウェスタンブロットは異なります。この技術は、標的生体分子の大きさについての情報を提供していないことに注意してください、そして単一のドットは2カ月あれば表示されます。異なるサイズのleculesが検出されます。最後に、間接ELISAを適切に定量化することができる信号を生成するために、リガンド結合アッセイのために使用されます。これは、mAbの特性のより多くの情報を提供し、それによってストリップの建設を促進します。 筋肉中のMbの濃度は、収集場所に応じて可変です。例えば、鯨類における水泳筋肉（軸の筋肉）は非水泳筋肉と比較しメガビットの有意に高い含有量を有し、そして若い鯨類からのサンプルは、動物の生活20を通じてMbの濃度が増加するため、より低いMbの濃度を持っているでしょう。当初、唯一の鯨類やシールのMbのをキャプチャCSF1H13は、コロイド金標識および抗体を検出することを意図していた、と多くの種のMbのを認識しCGF5H9は、テストライン上の捕捉抗体であろう。私たちは、検出抗体がより具体的であることを仮定し、捕捉抗体はすべきより一般的です。低Mbの濃度を有する鯨サンプルを使用した場合しかし、弱い陽性シグナルは、テストライン上で発見された（データは示さず）。代表的な結果で説明したように、2つのmAbの位置が逆転したときに問題が解決されました。良好な信号であっても新生児鯨類（本鎖ツノシマクジラ）（ 図4）のために示されました。 mAbの特性および濃度は、この現象に寄与するかどうかは不明です。 この研究では、PBSを用いて均質化凍結融解筋肉サンプルは、ストリップ試験で使用しました。他のサンプル状態と調製方法は、結果に影響を与え得ます。たとえば、Mbのような塩溶性タンパク質を純水ではなく、PBSを用いて抽出する必要があります。そうでなければ、抽出は異常な結果を招く可能性があり、不十分な場合があります。適切な抽出バッファー：肉のサンプル比は、ストリップ結果の成功の解釈のために部分的に責任があります。大amou対照試料（ 例えば、家畜）のヌクレオチド（1ミリリットルバッファ内の0.3グラム）が陽性の結果とぼやけた背景を引き起こす可能性があります。しかし、（1：0.03）この研究で使用割合は、正しい結果が得られました。唯一の新鮮な筋肉サンプルは、このストリップテストのために使用することができます。タンパク質を加水分解または他の動物からの鯨類の筋肉のサンプルもサンプルのためだけでなく、肯定的な結果を引き起こす可能性があり、（そのような醤油と沸騰による硬化など）特定の処理の後に変性することができた（データは示さず）。したがって、可変サンプル・ソースと異なる構造設計計画はストリップの開発中に使用されるべきであることが示唆されています。 結論として、このプロトコルは、鯨類のMBに強く反応する2 mAbの開発を説明し、これらのmAbは、シールや他の動物から鯨類のMbのを区別するために簡単なテストストリップに適用されます。鯨肉を同定するための信頼性のあるPCRベースのDNA分析は、利用可能な12であるが、それは私sの労働集約的で、時間がかかります。簡単なテストストリップは、規制機関21のために非常に望ましい鯨肉を識別するためにフィールドで使用することができ、信頼性と迅速な手法です。ストリップは、ウマまたはブタなどの動物の特定のMBを検出するために開発することができる可能性があります。

Historically, cetacean meat has been consumed in many parts of the world and this consumption continues today1. Due to the trophic level of cetaceans, high levels of mercury and other toxins are known to be present in their meat2. Therefore, the consumption of cetacean meat could lead to a health problem not only for high-risk groups such as pregnant women but also for the general population3. Furthermore, the contamination of cetacean meat with zoonotic or potentially zoonotic pathogens can also occur during its processing and storage4. It is difficult even for experienced agents to identify cetacean meats by their appearance alone. Therefore, a reliable scientific method of identification is required to differentiate cetacean meat from other meats. This would help to limit the consumption of cetacean meat.
Current methods of species identification include molecular techniques and immunological methods. Molecular techniques, such as polymerase chain reaction (PCR) and DNA sequencing, can be used to identify samples not only from raw meat5 and decomposed samples6 but also from processed foods such as cooked sausage and feedstuffs7,8. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), are commonly applied in food production to detect the meat content of, for example, pork9, beef10 and catfish11. PCR-based DNA analysis for the identification of cetacean meat is available12, and has helped prevent the illegal international trade of cetacean meat in Japan, South Korea, the Philippines, Taiwan, Hong Kong, Russia, Norway, and the United States1. These methods are effective and reliable, but they can take hours or days to complete and involve laborious steps. The identification of cetacean meats is usually based on molecular techniques and there is currently no immunological method available. For regulatory agencies, it is highly desirable to develop a dependable and rapid technique that can be used in the field to identify cetacean meats. 
Immunochromatographic strips are used as detection tools with the advantage of producing rapid result via a simple protocol that is suitable for use in the field. The principles of the immunochromatographic strip and ELISA are very similar, and includes antibodies, antigens and labels. Many different labels such as colloidal gold, carbon and latex have been used in the development of immunochromatographic strips. At present, this method is commonly used for detecting antibiotics, toxin, bacteria and viruses13, but it is rarely used for identifying proteins in meat14,15. Here we propose a lateral-flow chromatographic enzyme immunoassay for rapid detection of cetacean myoglobin (Mb).

<table border="0" cellpadding="0" cellspacing="0" width="1007">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">Phosphate buffered saline</td>
			<td style="width:183px;">AMRESCO</td>
			<td style="width:148px;">J373</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">Protein G HP SpinTrap&#160;</td>
			<td>GE&#160;Healthcare</td>
			<td>28-9031-34</td>
			<td>spin column containing Protein G Sepharose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">IsoStrip Mouse Monoclonal Antibody Isotyping Kit</td>
			<td>Roche</td>
			<td>11493027001</td>
			<td>Isotyping Strips, precoated with subclass- and light-chain-specific anti-mouse-Ig antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini Trans-Blot</td>
			<td>Bio-Rad</td>
			<td>170-3935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>Whatman</td>
			<td>Z613630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody blocker solution&#160;</td>
			<td>LTK BioLaboratories</td>
			<td style="width:148px;"></td>
			<td>To minimize nonspecific binding interactions of nonspecific IgG in the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">BCIP/NBT phosphatase substrate&#160;</td>
			<td>KPL</td>
			<td>50-81-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Detector HRP Microwell Kit, Anti-Mouse</td>
			<td style="width:183px;">KPL</td>
			<td>54-62-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Immunoplate MaxiSorp ELISA plate</td>
			<td>Thermo&#160;Fisher Scientific</td>
			<td>EW-01928-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiskan&#160;EX ELISA reader&#160;</td>
			<td>Thermo&#160;Electron&#160;Corporation</td>
			<td>51118170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colloid gold (40 nm) solution&#160;</td>
			<td>REGA biotechnology Inc.</td>
			<td style="width:148px;"></td>
			<td>40-50 nm is appropriate for immunostrip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Gibco</td>
			<td>15561-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapid test immno-strip printer</td>
			<td>REGA biotechnology Inc.</td>
			<td>AGISMART RP-1000&#160;</td>
			<td>Only suited for small scale production of immunostrips for research and development purposes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:512px;">Strip components (NC membranes, sample pads (#33 glass, S&#38;S), conjugate pads (#16S, S&#38;S) and absorbent pads (CF6, Whatman))</td>
			<td>REGA biotechnology Inc.</td>
			<td style="width:148px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freund&#8217;s adjuvant and incomplete Freund&#8217;s adjuvant&#160;</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:148px;">F5881, F5506</td>
			<td>Used to produce water-in-oil emulsions of immunogens</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:512px;">Acrylamide, gel buffer, ammonium persulfate (APS), tetramethylethylenediamine (TEMED)&#160;</td>
			<td style="width:183px;">Protech</td>
			<td style="width:148px;"></td>
			<td>Gel preparation for SDS-PAGE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coomassie brilliant blue R-250</td>
			<td style="width:183px;">Bio-Rad</td>
			<td style="width:148px;">1610436</td>
			<td>Protein staining in SDS-PAGE gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laemmli sample buffer and &#946;-mercaptoethanol</td>
			<td style="width:183px;">Bio-Rad</td>
			<td style="width:148px;">1610737, 1610710</td>
			<td>Dilute protein samples before loading on SDS-PAGE gels</td>
		</tr>
	</tbody>
</table>

development of a colloidal gold-based immunochromatographic test strip for detection of cetacean myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

モノクローナル抗体の特性 私たちは、鯨類のMBの、それぞれ2合成ペプチド（MKASEDLKKHGNTVLCとAIIHVLHSRHPAEFGC）を、認識2のIgG 1モノクローナル抗体（CGF5H9とCSF1H13）を開発し、これらはクジラMbのを迅速に検出するためのサンドイッチ型金コロイドイムノクロマト法テストストリップを構築するために使用しました。 図2は CGF5H9はおおよそ17キロダルトンの予測分子量で、単一の染色バンドとして鯨類および他の哺乳動物を検出することを示しています。ミンククジラ（ ミンクacutorostrata）は、他のクジラ類のバンドよりも比較的暗いバンドを示しています。約50キロダルトンのバンドが豚のために観察されるのに対し、バンドは、マグロ、鶏やシールのために存在しません。豚やマグロのための複数の非特異的なバンドがありますが、それはおおよそ17 kの予想分子量のバンドとしての鯨類やアザラシにのみ反応するため、CSF1H13は、非常に特異的ですダ。ミンククジラは、唯一の1時25分で観察された無信号で（データは示していない）1時10分に強いシグナルを示している。 図2は、ドットブロットで同一の結果を示しているクジラ。 図3Aは、CGF5H9は鯨類、ウサギ、イヌ、ヤギ、および牛（OD値&gt; 3.0）のための陽性シグナルを示していることを示しています。豚のための弱い正のシグナル（OD値= 1.5）。シール、鶏やマグロのための負の信号。 図3Bは CSF1H13は、鯨類とシール（OD値&gt; 3.0）の方に高い親和性を発現することを示しています。 CSF1H13とCGF5H9の両方がすべての4つの鯨類と強く反応することができます。多様な鯨類に広い反応性を示し、これらの4種が異なるファミリー（ネズミイルカ科：;：マイルカ;：;ドワーフマッコウクジラスナメリKogiidaeバンドウイルカナガスクジラ科ミンククジラ）からのものです。 ストリップ構築のために、CGF5H9は、トンを認識する彼鯨類および他の哺乳動物のMBS、コロイド金標識され、ミオグロビンに結合する検出抗体として使用し、CSF1H13のみ鯨類及びシールのMBを捕捉するために、テストライン上にコーティングされています。したがって、テストラインは、両方のmAbが鯨類Mbの検出正の信号を表示するように設計されています。非鯨類動物からMbが唯一のこれらの二つのmAbのいずれかによって検出することができるので、検査ラインは、他の動物からの筋肉試料を試験する陰性の結果を示しています。ウサギ抗マウスIgGコロイド金標識CGF5H9に結合するための制御線は、常に肯定的な結果を示しています。コントロールラインで失敗した結果は、ストリップ上の材料の品質が劣悪であることを示しています。 ストリップテスト 鯨類筋肉試料を使用した場合、図4は、テストラインおよびコントロールラインの双方の信号帯域を示す図です。サンプルは鯨類からのものではない場合には、制御線のwiで唯一の単一のバンドがありますテストラインでのバンドの不在番目。成功した結果を、10mlのPBSは、プラスチックまたは竹の棒を使用して、0.1％BSAを含有する混合物中にストリップを浸漬して筋肉0.03gの均質化した後、5〜10分で直接観察することができます。 15鯨類と三連で8非鯨類をテストした後、特異性（非鯨類のサンプルの割合が正しく識別）と感度（正しく識別鯨類標本の割合）は、両方の100％です。 <img alt="図1" src="/files/ftp_upload/53433/53433fig1.jpg" /> 免疫ストリップの 図1. デザイン。彼らが重なるようにすべてのコンポーネントはプラスチック製のバッキングカード上に慎重に積層されています。これは、試薬およびサンプルが膜を通過して吸収パッドまで流れることができます。 T：テストゾーン。 C：コントロールゾーン。この図は、ロー、C. らから再生されました。迅速な免疫金コロイドストリップ鯨類の肉抑制のための違法取引と消費：保全と公衆衛生のための含意するPLoS ONE 8、e60704（2013）。 DOI：10.1371 / journal.pone.0060704 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53433/53433fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53433/53433fig2.jpg" /> 図2ハイブリドーマ上清を用いたウェスタンブロットおよびドットブロット分析（A）CGF5H9、（B）CSF1H13。両方のハイブリドーマ上清は、おおよそ17キロダルトンの予測分子量で、単一の染色バンドとして鯨類を検出することができます。 MW：ミンククジラ、BND：バンドウイルカ、DSW：ドワーフマッコウクジラ、FP：スナメリ、PKW：ユメゴンドウ、N：PBS（ネガティブコントロール）。この図は、ロー、C. らから再生されている。cに対する迅速な免疫金コロイドストリップetacean肉拘束違法取引と消費：保全と公衆衛生のための含意するPLoS ONE 8、e60704（2013）。 DOI：10.1371 / journal.pone.0060704 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53433/53433fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53433/53433fig3.jpg" /> 図3. 間接精製したモノクローナル抗体を用いて、異なる種の筋肉抽出物のELISA。（A）CGF5H9、（B）CSF1H13。唯一の鯨類は、両方のmAbに強い陽性シグナルを生成することができます。この図は、ロー、C. らから再生されたクジラ肉拘束違法取引と消費のための迅速な免疫金コロイドストリップ：保全と公衆衛生のための含意するPLoS ONE 8、e60704（2013）。 DOI：10.1371 / journal.pone.0060704。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53433/53433fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53433/53433fig4.jpg" /> 免疫金コロイドストリップの 図4. 特異T：テストライン。 C：コントロールライン。鯨類筋肉試料が使用される場合にのみ、成功した結果（テストラインおよびコントロールラインの双方の信号帯域）を観察することができます。 （A）非鯨類のサンプル：1：牛。 2：ヤギ。 3：豚。 4：犬。 5：ウサギ。 6：マグロ。 7：チキン。 8：ハーバーシール（ ゴマフアザラシ属のvitulina）。 （B）日本鯨類のサンプル：1：ミンククジラ（ ミンクacutorostrata）。 2：ツノシマクジラ（ ミンクomurai）。 3：ハンドウイルカ（ ハンドウイルカ属のaduncus）。 4：イルカ（T.のtruncatus）。 5：サラワクイルカ（Lagenodelphis法政大学&lt;/ em&gt;の）。 6：インド太平洋のザトウクジライルカ（ スーザキネンシス ）。 7：ハナゴンドウ（ ハナゴンドウ ）。 8：Pantropicalはイルカ（Stenellaのattenuata）を見つけました。 9：シワハイルカ（ ステノbredanensis）。 10：ピグミーのシャチ（Feresa attenuata）。 11：コビレゴンドウクジラ（Globicephalaのmacrorhynchus）。 12：カズハゴンドウ（Peponocephalaエレクトラ ）。 13：ドワーフマッコウクジラ（Kogiaシマ ）。 14：ピグミーマッコウクジラ（K.のbreviceps）。 15：スナメリ（Neophocaenaのphocaenoides）。この図は、ロー、C. らから再生されたクジラ肉拘束違法取引と消費のための迅速な免疫金コロイドストリップ：保全と公衆衛生のための含意するPLoS ONE 8、e60704（2013）。 DOI：10.1371 / journal.pone.0060704 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53433/53433fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 鯨類 </td><td> 非鯨類 </td></tr><tr><td>ピグミーのマッコウクジラ（Kogiaのbreviceps） </td><td>ハーバーシール（ ゴマフアザラシ属のvitulina） </td></tr><tr><td>ドワーフマッコウクジラ（Kogiaシマ ） </td><td>犬（ オオカミfamiliaris） </td></tr><tr><td>コビレゴンドウクジラ（Globicephalaのmacrorhynchus） </td><td>ウサギ（Oryctolagusのカイウサギ ） </td></tr><tr><td>カズハゴンドウ（Peponocephalaエレクトラ ） </td><td>豚（ イノシシ ） </td></tr><tr><td>ピグミーのシャチ（Feresa attenuata） </td><td>ヤギ（ ヒマラヤ山羊 ） </td></tr><tr><td> Pantropicalは、（Stenella attenuata）イルカを発見しました</td><td>牛（Bosの牡牛座 ） </td></tr><tr><td>バンドウイルカ（ 火rsiops truncatus） </td><td>チキン（ ガルスガルス ） </td></tr><tr><td>イルカ（ ハンドウイルカ属のaduncus） </td><td>キハダマグロ（ キハダマグロ ） </td></tr><tr><td>サラワクイルカ（Lagenodelphis法政大学 ） </td><td></td></tr><tr><td>インド太平洋のザトウクジライルカ（ スーザキネンシス ） </td><td></td></tr><tr><td>シワハイルカ（ ステノbredanensis） </td><td></td></tr><tr><td>ハナゴンドウ（ グランパスグリセウス ） </td><td></td></tr><tr><td>スナメリ（Neophocaenaのphocaenoides） </td><td></td></tr><tr><td>ミンククジラ（ ミンクacutorostrata） </td><td></td></tr><tr><td>ツノシマクジラ（ ミンク大村 ⅰ） </td><td></td></tr></tbody></table> 筋肉はコレだったから種1.表この研究でCTEDとテストされています。種はマグロ、鶏肉、シール、陸生哺乳類の5種、および鯨類（4家族）の15種を含みます。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>種</td><td>ノーアク。 </td></tr><tr><td>ミンククジラ（ ミンクacutorostrata） </td><td> P02179 </td></tr><tr><td>ピグミーニタリクジラ（ ミンクedeni） </td><td> Q0KIY2 </td></tr><tr><td>ザトウクジラ（ ザトウクジラ ） </td><td> P02178 </td></tr><tr><td>コククジラ（ コククジラ ） </td><td> P02177 </td></tr><tr><td>マッコウクジラ（ マッコウクジラ属の大頭 ） </td><td> P02185 </td></tr><tr><td>ピグミーのマッコウクジラ（Kogiaのbreviceps） </td><td> Q0KIY5 </td></tr><tr><td>ドワーフマッコウクジラ（Kogiaシマ ） </td><td> P02184 </td></tr><tr><td>短期beakeD共通のイルカ（ マイルカ ） </td><td> P68276 </td></tr><tr><td>ヒレナガゴンドウ（GlobicephalaのMELAS） </td><td> P02174 </td></tr><tr><td>シャチ（ 鯱、シャチ ） </td><td> P02173 </td></tr><tr><td>カズハゴンドウ（Peponocephalaエレクトラ ） </td><td> Q0KIY3 </td></tr><tr><td> Pantropicalは、（Stenella attenuata）イルカを発見しました</td><td> Q0KIY6 </td></tr><tr><td>ハンドウイルカ（ バンドウイルカ ） </td><td> P68279 </td></tr><tr><td>ネズミイルカ（ ネズミイルカ ） </td><td> P68278 </td></tr><tr><td>アマゾンカワイルカ（ アマゾンカワイルカ ） </td><td> P02181 </td></tr><tr><td>ロングマンのオオギハクジラ（Indopacetusのpacificus） </td><td> Q0KIY9 </td></tr><tr><td>ハッブスオウギハクジラ（Mesoplodon carlhubbsi） </td><td> P02183 </td></tr><tr><td>キュビエ歯クジラ（ 紫phius cavirostris） </td><td> P02182 </td></tr><tr><td>ハーバーシール（ ゴマフアザラシ属のvitulina） </td><td> P68080 </td></tr><tr><td>牛（Bosの牡牛座 ） </td><td> P02192 </td></tr><tr><td>ヤギ（ ヒマラヤ山羊 ） </td><td> B7U9B5.3 </td></tr><tr><td>馬（ ウマ ） </td><td> P68082 </td></tr><tr><td>豚（ イノシシ ） </td><td> P02189 </td></tr><tr><td>犬（ オオカミfamiliaris） </td><td> P63113 </td></tr><tr><td>チキン（ ガルスガルス ） </td><td> P02197 </td></tr><tr><td>オーストリッチ（ ダチョウ ） </td><td> P85077 </td></tr><tr><td>キハダマグロ（ キハダマグロ ） </td><td> P02205 </td></tr></tbody></table> それぞれGenBankアクセッション番号を有する本研究で使用し、表2ミオグロビン配列は、種は、マグロを含みます鶏、ダチョウ、家畜哺乳類、シールと鯨類の18種（7家族）。このテーブルはロー、C. らから再生されたクジラ肉拘束違法取引と消費のための迅速な免疫金コロイドストリップ：保全と公衆衛生PLoSのONE 8のための含意、e60704（2013）。 DOI：10.1371 / journal.pone.0060704。

Watch this Scientific Journal Video about Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin at JoVE.com

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

クジラミオグロビンの検出のための金コロイドベースのイムノクロマト法テストストリップの開発

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

development-colloidal-gold-based-immunochromatographic-test-strip-for

Nanyang Technological University

Research

JoVE Journal

Biology

14.1K ビュー.  National Chiayi University. This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals. 

ビデオ: Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

ニューロンとグリアの開発研究ラットの脳の集計文化の調製

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

ウェスタンブロッティング：サンプル調製を検出する

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 &mu;g ml-1 down to 10 ml of 198.7 &mu;g ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 &mu;g ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 &mu;g ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 &mu;g ml-1 down to 4 ml of 8,539.9 &mu;g ml-1) with minimal aggregation.

タンジェンシャルフロー限外濾過：コロイダルシルバーナノ粒子の大きさの選択と集中のための &quot;グリーン&quot;メソッド

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

胚発生時に細胞運命のコミットメントをテストするために割球植

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

ライブの検出<em&gt;エシェリヒア·コリ</em&gt; O157：PMA-qPCRによりH7細胞

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...