著者は、リサーチ＆コラボレーション（SPARC）賞小薬局·アワードを通じて、このプロジェクトの財政支援のために薬局のプレスビテリアンカレッジスクールの研究局に感謝したいと思います。著者は、ジョン·Smink、水生動物研究所のマネージャー、クレムソン大学、クレムソン、彼の技術支援の原稿の改訂のためのSCに感謝。

注：これらのプロトコルは、プレスビテリアンカレッジのIACUCのガイドラインに従ってください。 表1に列挙した材料を取得します。 2.メーカーの指示に従って5シェルフChromeを組み立てます。これは施設を収容するために使用されます。 3.水リザーバ及び濾過システム（図1A及び図2）を構築する。 <ol><li>ガイドとして、図1Aを使用して、最終的な設置場所とサイズを決定するために、18ガロンのバケツ、ポンプ、フィルター、紫外線ランプをレイアウト。 </li><li>部品がレイアウトされると、測定し、組み立てに必要な長さに¾ &quot;PVCパイプをカット。特定のスペースのニーズと制限に応じてすべてのパイプの長さを変更します。 </li><li> PVCプライマーとセメントを使用して、以前にレイアウトとして配管や継手を組み立て、以下の（3.3.2および3.3.3）を参照してください。 <ol><li>サンドサンドペーパーによるパイプの切断端部は、タッチANに滑らかになるまでデブリがないdは。 </li><li>総理パイプの外側と塩ビプライマーから含ま綿棒で購入したPVCプライマーとフィッティングの内側。 </li><li>含ま綿棒を使用してパイプの外側にPVCセメントを適用することにより、一緒にパイプと継手セメント。塩ビセメントを必要とするすべての手順について繰り返します。 </li><li>棚建設の片側に18ガロンのバケツを置きます。 </li><li>リザーバタンクの側面にバルクヘッドコネクタを取り付けます。 </li><li>バルクヘッドに¾ &quot;PVCパイプを取り付けます。 </li><li> ¾ &quot;¾を使用してポンプ入口へのパイプ」のもう一方の端を取り付け金具作るネジ付き。 </li><li> 2 90度の肘と¾ &quot;PVCパイプを使用してポンプにフィルタを接続します。 <ol><li> 1活性炭フィルター、続いて1枚のフィルタがデブリをフィルタし、水に塩素および化学のレベルを減少させるために使用する。 </li></ol></li><li>フィルタaccordiにUVランプを接続します製造元の説明書をngの。 </li></ol></li><li>ワイヤータイを使用して、ラックの最初の棚の上部側にポンプとフィルターシステムを取り付けます。選択した棚/ラックのサイズとレイアウトによるアセンブリおよび添付ファイルの順序を変更します。 </li><li>ポンプとフィルターシステムが組み立てられ、棚に取り付けられた後、水の貯水池への水のガロン当たり1 biobal​​lを追加します。 </li></ol> 4.主電源ライン（図1Bおよび2）を構築する。 <ol><li>測定主電源にUVランプを接続するPVCパイプ」3/4部分を切断し</li><li> UVフィルターから来るのPVC排気」¾にティーを接続することにより、主電源ラインを開始するためのPVCティー」¾を使用してください。 。 <ol><li>システムのメンテナンス洗浄のためのドレインとして機能するPVCティーの底に開閉弁を追加します。 </li><li> 「¾を測定し、切断したPVCティー」3/4上部から主電源ラインを起動し、PVCラックの棚のそれぞれについて、供給管を提供する所望の長さのパイプ。 </li><li>インストール¾ &quot;ラックの各棚に入ります水平線のためのPVCティーを。 </li><li>必要に応じて（提示）、追加¾ &quot;開閉弁各棚供給ライン各棚の単離およびシステムのモジュール設計を可能にする。 </li></ol></li><li>一番上の棚に線を減圧として¾ &quot;PVCパイプを使用してください。この行が棚の裏に実行されている場合は、保存のために、この棚を使用しています。開閉弁¾ &quot;メイン排気ライン（ 図1B）の線減圧を終了する。 </li><li>上記のステップは、プライム完了し、リザーバとろ過システムを構築する際に、以前に行ったように一緒に塩ビ管を固めるされたら。 </li></ol> 5.主排気ライン（図1Cおよび2）を構築します。 <ol><li>ラックfの他方側に主排気ラインの垂直方向の長さを実行する主供給ラインにデキュー。各棚から魚の水槽からトップとドレインラインでの主電源ラインを受け取るために、この垂直線のために2 &quot;PVCパイプを使用してください。 </li><li> 「垂直主排気ライン用の塩ビ管と2を使用する「2の部分をカットし、各棚から水槽のドレイン線を受け取るためにPVCティーを。スポンジフィルター上で再生水を排出するストックタンク内の主排気ラインを終了します。 </li><li> 2「PVCパイプ片の全長を開いて1インチをカットし、水槽からの水平ドレイン線用のPVCティー垂直主排気ラインに接続する。 </li><li> PVCキャップ2」2水平ドレイン線を「キャップ。 </li><li>首相と必要な付属品（コネクタ、乳首や肘）を使用して作品を固める。 </li></ol> 6.水槽（図3）を構築。 <ol><li>ドリルで、ホールソーと上辺以下の「魚タンク1の背面に穴 &quot;1 1/8を切った。 </li><lI&gt;（外部へのタンクの内側から）穴を介して（添付＃18 Oリング付き）のねじ¾ &quot;PVCオスアダプタをスライドさせます。 </li><li> 90度を取り付け、締め3/4「魚タンク排気管を作成するために男性のアダプタにPVC肘をスレッド化。排気システムを通過するゼブラフィッシュを防ぐために、保持ナットをねじ込む前に、細かいメッシュの部分を追加します。 </li><li>その排気管がタンクの後ろに排水ラインのオープンスペースに終わると水槽を置き</li></ol> 7.水システムの起動<ol><li>そのようなリザーバーに使用されるものなど、いくつかの容器中に、システム内に含まれる水の3倍の量を測定する。この水は48時間調整のため室温で放置しましょう​​。 </li><li>システムに水を追加し、3日間の最低循環してみましょう。 <ol><li>汚染物質のTHAのシステムを取り除くために、循環の最初の3日間を通して積極的にシステム内の水を変更tは、パイプ内に存在する。 </li></ol></li><li>スターターゼブラフィッシュを追加し、窒素サイクルの確立を取得することができます。 1.8 Lあたり7ゼブラフィッシュを保持し、かつのLiNbO 4とマクナブ6によって示唆されているように、週末の間、1日1回、週の日の間に1日2回、それらを供給し、一貫性のpHを7に正規化する。 </li><li> 、硝酸塩（&lt;75 mg / Lの）を監視魚を追加した後の最初の週の間の毎日の亜硝酸塩（&lt;10 mg / Lの）、およびアンモニウム（0ミリグラム/ L）、週に2回、一度所望のレベルは、通常の水の変更に維持されている。 </li></ol>

<ol>
	<li>Detrich, H., Westerfield, M., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Cellular and Developmental Biology, Part A.. , (2010).</li><li>Grim, J. 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=Hemogen+(hemgn),+a+transcription+factor+that+regulates+erythropoiesis+and+skeletogenesis+in+zebrafish.">Hemogen (hemgn), a transcription factor that regulates erythropoiesis and skeletogenesis in zebrafish.</a> , (2013).</li><li>Kim, S., Carlson, R., Zafreen, L., Rajpurohit, S. K., Jagadeeswaran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular,+easy-to-assemble,+low-cost+facility.">Modular, easy-to-assemble, low-cost facility.</a> Zebrafish. 6 (3), 269-274 (2009).</li><li>Linbo, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+(Danio+rerio)+husbandry+and+colony+maintenance+at+the+Northwest+Fisheries+Science+Center.">Zebrafish (Danio rerio) husbandry and colony maintenance at the Northwest Fisheries Science Center.</a> U.S. Dept. Commer., NOAA Tech. Memo. 62, (2009).</li><li>Avdesh, 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. 69, 4196 (2012).</li><li>McNabb, A., Scott, K., Ochsenstein, E., Seufert, K., Carl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+be+afraid+to+set+up+your+fish+facility.">Don't be afraid to set up your fish facility.</a> Zebrafish. 9 (3), 120-125 (2012).</li><li>Garcia, R. L., Sanders, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+cleaning+and+disinfection+procedures+in+a+zebrafish+rerio)+facility.">Efficacy of cleaning and disinfection procedures in a zebrafish rerio) facility.</a> J Am Assoc Lab Anim Sci. 50 (6), 895-900 (2011).</li><li>Rivas-Boyero, 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=Pharmacological+characterization+of+a+nociceptin+receptor+from+zebrafish+(Danio+rerio).">Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio).</a> J Mol Endocrinol. 46 (2), 111-123 (2011).</li><li>Sanchez-Simon, F. M., Zhang, X. X., Loh, H. H., Law, P. Y., Rodriguez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+regulates+dopaminergic+neuron+differentiation+via+miR-133b.">Morphine regulates dopaminergic neuron differentiation via miR-133b.</a> Mol Pharmacol. 78 (5), 935-942 (2010).</li><li>Velasco, E. M., Law, P. Y., Rodr&#237;guez, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mu+opioid+from+the+zebrafish+exhibits+functional+characteristics+as+those+of+mammalian+mu+opioid+receptor.">Mu opioid from the zebrafish exhibits functional characteristics as those of mammalian mu opioid receptor.</a> Zebrafish. 6 (3), 259-268 (2009).</li></ol>

著者は、この論文のための目的の競合を宣言しません。

この記事の主な目的は、機能的なゼブラフィッシュ施設の建設が複雑ではなく、最小限の工事知識を持つ初心者の魚の研究者が構築できることを実証することである。さらに、この資料では、この機能のゼブラフィッシュの施設のための材料は、地元のペット用品店やホームセンターから容易にかつ低コストで取得できることを示している。すべての材料とツールは、システムの構築や魚の?...

彼らはシステム及び生物レベルで翻訳されるために、インビトロ研究に許可しているため、生体内でのモデルは、総合的な生物医学研究の実施に不可欠である。例えば、小型哺乳類、すなわちげっ歯類との研究は、薬物の開発および利用可能な薬物の改善に必要な証明されている。しかし、（財政的およびロジスティック）複雑さは、それは実験のために齧歯類の施設を建設し、維持することは事実上不可能小さな研究の学術機関で提案するかもしれない。それにもかかわらず、in vivoでの研究は、より少ない複雑な物流を必要とする他の動物種を用いて行うことができる。ゼブラフィッシュ（ ゼブラフィッシュ ） が正常に遺伝学、開発、病態生理、および薬理1,2,10,11,12を研究するためのin vivoモデルとして使用されてきた。 rに比較した場合の実験のためにゼブラフィッシュを収容するために必要な設備が比較的単純でかなり安価であるodent施設。 Kim ら 3は最近、詳細に部品や人件費の両方が含まれている約1500ドル、のために構築することができる80のタンクゼブラフィッシュの住宅システムを記載している。これは商業的供給者の任意の数から既製、80タンクシステムを購入することを要求され​​ることはほぼ8500ドルとは全く対照的である。しかしながら、説明は、Kim らによって与え。魚研究の初心者のために従うことが複雑で困難な場合がある。我々はゼブラフィッシュ施設が1500ドルの投資のための簡易的に構築することができると信じています。 小さ ​​な学術機関での生体内の施設の設立は、より強力かつ徹底した調査を生成すると追加のツール彼らの学部を提供しています。 中でサポートされているin vitroデータvivoでの結果は、一般的にのみ体外のアプローチで使用して得られたデータよりもより包括的な生物医学研究を生産する。そのため、in vivoでの研究施設の実装が大幅に全体的な研究努力における機関の成功強化します（研究への学生の暴露、出版物、通信、外部資金の追求、 等 。）。 この研究で説明するようなゼブラフィッシュ施設の建設は、フィナンシャル·タイムズを収縮時に（in vivoでの研究施設なしまたは機関）限られた施設との研究教育機関に最適です。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Item</td>
			<td>Brand</td>
			<td>Quantity</td>
			<td></td>
		</tr>
		<tr>
			<td>74-in H x 48-in W x 18-in D 5-shelf Steel Freestanding Shelving Unit</td>
			<td>Style Selections</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>18-Gallon General Bucket/Tote</td>
			<td>Centrex Plastics: LLC&#160;Rugged Tote</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Outlet Power Strip With 15-Ft. Cord</td>
			<td>Master Electrician</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Watt UV Water Garden Clarifier FiIter and UV lamp</td>
			<td>Smartpond&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole House Water Replacement Filter</td>
			<td>Whirlpool</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>Opaque Whole-House Pre-Filtration Housing</td>
			<td>Whirlpool</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread Seal Tape</td>
			<td>William H Harvey</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>8-fl oz LO-VOC PVC Cement</td>
			<td>Oatey&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>8-fl oz LO-VOC Primer</td>
			<td>Oatey&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-in x 100-ft Vinyl Drip Irrigation Distribution Tubing</td>
			<td>Mister Landscaper&#160;</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-in Barbed Drip Irrigation On/Off Valve</td>
			<td>Mister Landscaper</td>
			<td>33</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Port NPT Irrigation Manifold with Filter</td>
			<td>&#160;Mister Landscaper</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>Drop-In Filter with House&#160;</td>
			<td>Whirlpool</td>
			<td>6</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia 90-Degree PVC Elbow</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia 90-Degree Tee</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia 90-Degree Slip Elbow</td>
			<td>LASCO</td>
			<td>15</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Adapter</td>
			<td>LASCO</td>
			<td>7</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Tee</td>
			<td>LASCO</td>
			<td>12</td>
			<td></td>
		</tr>
		<tr>
			<td>2-in Dia PVC Cap</td>
			<td>LASCO</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>1-in Dia x 3/4-in Dia PVC Adapter</td>
			<td>LASCO</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in PVC Socket In-Line Ball Valve</td>
			<td>American Valve</td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2-in Dia x 3/4-in Dia PVC Adapter</td>
			<td>Lasco</td>
			<td>8</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4-in Dia PVC Cap</td>
			<td>Lasco</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>2-In x 5-Ft Pvc Pipe</td>
			<td>Charlotte Pipe</td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;-In x 10-Ft Pvc pipe</td>
			<td>Charlotte Pipe</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>11/16x1-7/80 PVC ring</td>
			<td>SXT</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; PVC male adapter</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;&#8221; PVC coupling&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189;&#8221; PVC female adapter&#160;</td>
			<td>SXT</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>link</td>
			<td></td>
		</tr>
		<tr>
			<td>1.8 L Dual Beta Keeper</td>
			<td>Top Fin</td>
			<td>27</td>
			<td></td>
		</tr>
		<tr>
			<td>&#190;&#8221; PVC male adapter (w/ #18 O-ring)</td>
			<td>SXT</td>
			<td>28</td>
			<td></td>
		</tr>
		<tr>
			<td>90-degree &#190;&#8221; threaded PVC elbow</td>
			<td>SXT</td>
			<td>27</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Mesh</td>
			<td>N/A</td>
			<td>1 yard</td>
			<td></td>
		</tr>
		<tr>
			<td>Bulkhead Connector</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hacksaw</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole saw</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable Wrench</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pliers&#160;</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape Measure</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety Glasses</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Work Gloves</td>
			<td>N/A</td>
			<td>1</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of an affordable and easy-to-build zebrafish facility

生体内では生物医学研究は、臨床の進歩に体外調査結果に変換するために極めて重要である。限られたリソースを持つ小型の学術機関は、それが事実上不可能に研究のための典型的なげっ歯類の施設を構築し、維持するために見つける。ゼブラフィッシュ研究は薬理学、生理学、発達および遺伝的研究におけるin vivo研究のための貴重な代替手段であることが実証されている。この記事では、機能的なゼブラフィッシュ施設が簡単かつ手頃な価格の方法で構築することができることを実証している。私たちは、そのような施設は、最小限のツールと​​専門知識を用いて約1営業日で構築することができることを実証している。本研究で構築し27 1.8 L水槽のゼブラフィッシュ設備の費用は約1500ドルだった。私たちは3ヶ月間の初期作業150魚のコロニーの維持が千ドルであると推定している。このプロジェクト研究のためにゼブラフィッシュの養殖するために導入された、関係の学生が、提案している。

ポンプとFltersシステムの構築 ラックを組み立てる最初のステップを設定したら、ポンプおよびフィルタシステムを構築することである。フィルタは、システムのポンプに接続されており、容易にフィルタのメンテナンスや交換を可能にする第1の棚の底レベルの上方に位置している。同様に、UVランプは、フィルタのPVCパイプに接続され、必要に応じて（ 図4A）のよう...

Watch this Scientific Journal Video about Construction of an Affordable and Easy-to-Build Zebrafish Facility at JoVE.com

Construction of an Affordable and Easy-to-Build Zebrafish Facility

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

手ごろな価格で簡単に構築ゼブラフィッシュ施設の建設

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

construction-of-an-affordable-and-easy-to-build-zebrafish-facility

Research

JoVE Journal

Biology

16.2K ビュー.  Presbyterian College School of Pharmacy. A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

ビデオ: Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

代謝標識とBioorthogonalクリックケミストリーによるゼブラフィッシュ胚でのイメージンググリカン

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

アダルトゼブラフィッシュ網膜にモルフォの in vivoエレクトロポレーションに

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

リジェネアダルトゼブラフィッシュテールフィンにMorpholinos の in vivoエレクトロポレーションで

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

ゼブラフィッシュの定期的ケアとメンテナンス（<em&gt;ゼブラフィッシュ</em&gt;）研究所：はじめに

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

高解像度メルト解析を用いたPCRを使用して、迅速かつ効率的なゼブラフィッシュ遺伝子型決定

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

アダルトゼブラフィッシュ側線再生のためのアッセイ

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

サブクローニングプラス挿入（SPI） - ジーンターゲティングベクターの迅速な構築のための新しい遺伝子改変方法

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...