体外受精と雌性発生二倍体のための方法は1970年代と1980年代にオレゴン大学のジョージStreisingerの研究室でシャーリーンウォーカーによるゼブラフィッシュのために開発されました。方法は、ここで説明ゼブラブック3で完全に利用可能であるシャーリーンのプロトコルから変更されていません。 体外受精と雌性発生二倍体のための方法は1970年代と1980年代にオレゴン大学のジョージStreisingerの研究室でシャーリーンウォーカーによるゼブラフィッシュのために開発されました。方法は、ここで説明ゼブラブック3で完全に利用可能であるシャーリーンのプロトコルから変更されていません。

初期の圧力の概要注：このようなtricaine、魚の水とハンクス生理食塩水、このプロトコルで使用される試薬の多くのためのレシピは、ゼブラフィッシュブック3（第10章をオンラインで利用することができます<a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> ） <ol><li> UV不活化精子を用いた体外受精で胚を作り出​​す。 </li><li>すぐに卵を水でいっぱいに充填されたガラスバイアル（プラスチック製のスナップキャップ付き）に受精卵を転送する。ラテックスゴムの薄いシートでバイアルの開口端部を覆い、ゴム製オーバーバイアルにトップを（穴が切断されているに）スナップ。 </li><li>油圧プレスでバイアルを置き、1.4分で受精した後、8000 psiの（多くのプレス機を2インチ径のピストンにかかる圧力を読み取るために較正されている、との読みがPSIに変換する必要があることに注意）に圧力を上げる</li><li>受精後6分で始まる、徐々に1分間の期間にわたって圧力を解放。 </li></ol>詳細な初期圧力の手順<ol><li> 実験の前日 <ol style="list-style-type:lower-alpha;"><li>午後遅くに、別々の男性と女性が圧迫される。彼は遺伝物質を寄与されることはありませんので、どんな歳の男性を使用することができます。雌は腹に大きなや脂肪になるはずです。男性は黄色と元気なはずです。 </li><li>それらは容易に午前中にアクセスすることができるタンクを保持するには別に一晩女性と男性を保持する。 </li></ol></li><li> 実験の朝 <ol style="list-style-type:lower-alpha;"><li>ハンクス溶液を調製、炭酸水素ナトリウムを加える。 </li><li>魚の施設へ移動：アイスバケット、ハンクス、Tricaine、タイマー、精子を収集するためのチューブ。 </li><li>魚の水にTricaineを薄める</li></ol></li><li> 精子を集める <ol style="list-style-type:lower-alpha;"><li>氷上ハンクス液の明確なバイアルを準備。測定〜男性当り50μlを圧迫する。 </li><li> tricaineに浸漬して、一度に3または4人の男性を麻酔。これらは、プラスチックスプーンで麻酔から持ち上げることができます。 </li><li>魚の水ですすぎ、ペーパータオル（非常に重要：水を活性化精子）に&quot;湿気、乾燥を&quot;汚点。 </li><li>落射照明と低倍率での実体顕微鏡下で泡のホルダーで魚をマウントします。鉗子と骨盤フィンを分離し、排出腔の開口部にマイクロキャピラリーを置きます。 </li><li>スムース（ミリポア）ピンセットで慎重に、しっかりと魚のストローク側面。 </li><li>乳白色の精子が生殖孔から出てくるように、穏やかな吸引を用いてマイクロキャピラリーでそれを収集する。 </li><li>氷冷ハンクス生理食塩水でいくつかの男性からのプールが精子。 50から10人の男性から精子の数百の卵の受精に適しています。寒いハンクスので精子は数時間あるいは数日のために効率的に卵を受精していきます。 &quot;眼球&quot;精子の濃度、濁った懸濁液を作るのに十分な収集。 </li></ol></li><li> UV不活性化精子 <ol style="list-style-type:lower-alpha;"><li>氷とガラスペトリ皿の底を埋める。 </li><li>皿に氷の上にwatchglassを置く。 </li><li>パスツールピペットを使用して、watchglassに試験管から精子を転送する。それと空気の気泡を取ることなく、できるだけピペットに精子をできるだけ多く使うようにしてください。あなたがピペットからそれらを追放しているとしても、watchglassでバブル精子をしないでください。時計皿上に薄くで精子を排出する。このピペットを捨てます。 </li><li> UVクロスリンカーにペトリ皿を置き、 </li><li> 2分間架橋剤をオンにする</li><li>清浄なピペットを使用して、watchglassから精子を取り出して、氷上でUV精子と場所マークのエッペンドルフチュー​​ブに移す。 </li></ol></li><li> 採卵と体外受精 <ol style="list-style-type:lower-alpha;"><li> tricaineで一度に3つまたは​​4つの女性を麻酔。 </li><li>魚の水ですすぎ、ペーパータオル上で&quot;生乾きにしたの&quot;汚点。余分な水は卵を膨張し、受精を防ぐことができます。 </li><li> 35mmプラスチックシャーレに湿った（ウェットではない）指で女性を置き、腹に慎重にしっかりと押してください。彼女が産卵のために用意されている場合、彼らは非常に簡単に出てくるでしょう。 </li><li>へらで卵を収集し、水にメスを返します。 （下記参照）女性に残っている卵は長すぎる白と水っぽくなるのに対し、良い卵は、黄色がかった、透明感のある色です。良い卵を得る保証するために、魚の施設内に来てライトの後の最初の2時間の間にそれらを収集する。乾燥アウトを防ぐために覆われて卵を保管してください。 </li><li>卵にUV精子懸濁液の30〜50μl加えて、ピペットの先端で軽くかき混ぜる。 </li><li>即時LYは、魚の水の約1 mlを追加し、タイマーを起動します 。この時点で開発するために左の卵は半数体となります。 </li></ol></li><li> 初期の圧力注：。AGは、受精後90秒以内に達成しなければならないステップ！ <ol style="list-style-type:lower-alpha;"><li>卵は水の添加によって活性化された時点でタイマーを起動します。 </li><li>しばらく待ってから、皿の中央に、より多くの水、スワール卵を追加。 </li><li>カットオフと洗練されたパスツールピペットを用いて、圧力のバイアルに受精卵を転送する。 </li><li>必要に応じて彼らはほとんどバイアルのリップで水のビーズを残して、あふれているように、バイアルに多くの卵の水を加える。 </li><li>水のビーズ上のゴムの上に置き、ゴム上のプラスチック製の蓋を固定します。 </li><li>補充シリンダーをするより多くの卵の水を加え、加圧シリンダーで上下逆さまにバイアル（s）を置きます。しっかりとシリンダーの上部を置く。 </li><li>プランジ​​ャーとプレスで円筒を置き、8000ポンド/平方を適用します。インチ</li><li>活性化の時間後の6分間（圧力の4.5分の合計時間）まで、プレスで円筒にしておきます。 </li><li>活性化後6分で、記者からシリンダーを削除し、シリンダーからバイアルを取り外してください。慎重に胚の冷却を防ぐためにバイアルを乾燥させる。 </li><li>シリンダーと補充卵を水でリセット。 </li><li>必要に応じて部分的に卵を水で埋めることによって、次のバイアルを準備。 </li><li>卵のすべてのバッチについて繰り返します。 </li></ol></li></ol>注：シリンダが一度に3バイアルまで保持されるので、すでにtricaineで他の雌は卵を与えるかどうかを確認するために数分を卵の受精を遅らせることをお勧めします。これは、一度に複数のバッチを処理することにより、最高速度のものを助ける。プレスが使用されている回も、治療が完了するまでtricaine内の任意のより多くの魚を入れていない覚えている。魚が長すぎる麻酔に滞在できるようにすることは、それらを殺すことができる。第二に、あなたがプレスが使用されている間に魚を絞る、と卵を得れば、彼らはマスコミを再び使用することができる時間で実行可能なように乾燥しすぎかもしれません。

<ol>
	<li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+analyses+and+gene-centromere+distances+in+zebrafish.">Segregation analyses and gene-centromere distances in zebrafish.</a> Genetics. 112 (2), 311-311 (1986).</li><li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-293 (1981).</li><li>Westerfield, M. <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 Book: A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Johnson, S. L., Africa, D., Horne, S., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-tetrad+analysis+in+zebrafish:+mapping+the+ros+mutation+and+the+centromere+of+linkage+group+I.">Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.</a> Genetics. 139 (4), 1727-1727 (1995).</li><li>Beattie, C. E., Raible, D. W., Henion, P. D., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+pressure+screens.">Early pressure screens.</a> Methods Cell Biol. 237 (71), 2575-25 (1999).</li><li>Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centromere-linkage+analysis+and+consolidation+of+the+zebrafish+genetic+map.">Centromere-linkage analysis and consolidation of the zebrafish genetic map.</a> Genetics. 142 (4), 1277-1277 (1996).</li><li>Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutants+with+disrupted+early+T-cell+and+thymus+development+identified+in+early+pressure+screen.">Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen.</a> Dev Dyn. 237 (9), 2575-2575 (1999).</li></ol>

この方法により製造された胚は、真の雌性発生二倍体であることを確認することが重要です。コントロールとして、通常の二倍体の卵（UV不活化せずに精子のいくつかを取っておくことにより）と一倍体の胚（EPの手順を経由せずにUV精子で受精卵のクラッチを脇に保つことによって）のクラッチのクラッチを生成する。 1日の後の受精の一倍体胚ではショートボディの軸、不規則な脳と体節?...

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<td>Tricaine</td>
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<td>Sigma</td>
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<td>Watch glass</td>
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<td>French Press</td>
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<td>Pressure cylinder</td>
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<td>Instant ocean</td>
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<td>Pasteur pipettes</td>
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<td>Cut off the narrow end, fire polish</td>
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<td>UV cross-linker</td>
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making gynogenetic diploid zebrafish by early pressure

二倍体真核生物におけるヘテロ接合性は、しばしば遺伝学的研究が面倒になります。ヘテロ接合の雌から直接実行可能なホモ接合二倍体の子孫を生成するメソッドは、F1変異女性が加速フォワード遺伝学的スクリーニングに有害な突然変異を直接スクリーニングすることができます。 Streisingerら。<sup&gt; 1,2</sup&gt;は、紫外線不活化精子とゼブラフィッシュ卵を活性化し、物理的処理（熱または圧力）そのdeploymerize微小管を使用して第二減数または最初の接合子の細胞分裂のどちらかを防ぐことによって、雌性発生（ホモ接合）二倍体のゼブラフィッシュを作製するための方法を説明した。 &quot;初期の圧力&quot;（EP）メソッドは、受精直後に起こる減数分裂IIを、ブロックします。 EP法は、いずれの性の肥沃な大人に開発することができます実行可能な胚の割合が高い生成します。方法は、減数分裂I.ホモ接合体の変異がセントロメア遺伝子座は50％とテロメア遺伝子座のための1％未満の間でEPのクラッチで検出された際に再結合することにより、動原体から分離されたものを除き、すべての遺伝子座でホモ接合である胚を生成します。このメソッドは、ゼブラフィッシュ帳からそのまま再現されている<sup&gt; 3</sup&gt;。

Watch this Scientific Journal Video about Making Gynogenetic Diploid Zebrafish by Early Pressure at JoVE.com

Making Gynogenetic Diploid Zebrafish by Early Pressure

これは、すぐに紫外線で不活化精子と受精した後、第二減数分裂をブロックすることにより、雌性発生二倍体のゼブラフィッシュの胚（遺伝のみ寄与母親由来胚）を生成するための方法です。 EPの胚は、最初の減数分裂時の組換えが原因で完全にホモ接合体ではない、しかし、彼らは再結合することにより、動原体から分離されていないすべての遺伝子座でホモ接合である。

初期の圧力による雌性発生二倍体のゼブラフィッシュを作成

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

making-gynogenetic-diploid-zebrafish-by-early-pressure

Research

JoVE Journal

Biology

13.4K ビュー.  University of Oregon. これは、すぐに紫外線で不活化精子と受精した後、第二減数分裂をブロックすることにより、雌性発生二倍体のゼブラフィッシュの胚（遺伝のみ寄与母親由来胚）を生成するための方法です。 EPの胚は、最初の減数分裂時の組換えが原因で完全にホモ接合体ではない、しかし、彼らは再結合することにより、動原体から分離されていないすべての遺伝子?...

ビデオ: Making Gynogenetic Diploid Zebrafish by Early Pressure

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

小児神経イメージングプロトコル、ガイドラインと手順 - MRイメージングチャイルドプレイを作る

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植によるキメラゼブラフィッシュ胚の生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

LAESI質量分析による生体組織とバイオフィルムの大気圧分子イメージング

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

大気圧質量分析法による単一細胞の直接分析

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

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 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

半数ゼブラフィッシュ胚の生成により<em&gt;体外</em&gt;受精

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...