サポートのおかげで可能に作られたこの作品: パヴィア大学ドイツ連邦共和国 2016;パルマ大学フィル 2014、2016;本研究の遂行に必要なプラスチック製品を供給するためのキネシス。博士シェーン ウィンザー (工学部、ブリストル大学、イギリス) は、Cell_PIV ソフトウェアを提供することを感謝いたします。

動物を含むすべてのプロシージャは、制度的動物のケアし使用とパヴィア大学の倫理委員会によって承認されました。動物は、22 ° C、湿度 60% の空気と 12:12 時間の明暗サイクルの条件の下で維持されました。
1. 卵巣の分離
<ol><li>腹腔内 2 4-11 週齢 CD1 雌マウス 10 U の卵胞刺激ホルモン滅菌 1 mL インスリン注射器を注入します。</li>
	<li>46-48 h を待ちます。</li>
	<li>マウスの重さし、Zoletil (Tiletamina、Zolazepan cloridrate) の 50 mg/kg の筋肉内注射で麻酔。頚部転位によって安楽死させます。</li>
	<li>剥離鉗子のペアを使用して腹壁を覆っている皮膚を把握します。はさみで皮膚と体壁をカットし、鉗子で切開を開けます。</li>
	<li>ピンセットのペアを使用して、移動勇気はさておき、正確な子宮角と四肢の運動で卵巣をローカライズします。</li>
	<li>時計鉗子を用いた卵巣優しく押しながら子宮にその靭帯をカットするはさみを使用します。他の卵巣を繰り返します。</li>
	<li>37 ° c、5% CO2空気中で加温 M2 分離培地 1 mL を含む 35 mm 細胞培養シャーレに収集した卵巣を転送します。</li>
	<li>脂肪と顕微鏡下で高級はさみを使用して卵管を取り外します。</li>
</ol>2. 卵丘卵子複合体の単離
<ol><li>幽門洞卵丘卵子複合体 (Coc) が受動的に解放されるまで、滅菌ピンセットや 1 G 滅菌インスリン針を使用して卵巣の表面を穴をあけます。</li>
	<li>卵丘細胞口制御の手を引っ張ってパスツール ピペット (直径 200 μ m) を使用しての 3 つ以上の層で Coc を収集し、培 M2 300 μ L ドロップにそれらを転送します。</li>
	<li>数秒間に軽くピペッティングして手を引いたパスツール ピペット (80 μ m) を使用して卵母細胞を囲む積雲セルを削除します。</li>
	<li>繰り返し卵母細胞から転送 M2 中の 20 μ L ドロップを別卵丘細胞が完全に除去されるまで。</li>
</ol>3. クロマチン組織ベースの卵母細胞の分類
<ol><li>最終濃度 5 mg/mL の 1x PBS で希釈の母液から M2 を中で 0.05 μ G/ml ヘキスト 33342 染色液を準備します。</li>
	<li>ヘキスト染色液 35 mm シャーレの蓋の下部の 3.5 μ の微小液滴を配置します。</li>
	<li>ドロップを染色ヘキストにそれぞれの単一の卵を転送、予め温めておいた (37 ° C) 加熱プレートの上に皿を置き、光博覧会を避けるために暗いふた付きカバーします。</li>
	<li>常温静置後の 10 分は後より 1 〜 2 秒の紫外線 (330-385 nm) の下で 10 倍の倍率で蛍光顕微鏡で卵母細胞を観察します。</li>
	<li>ヘキスト正クロマチン核小体を周囲のリングを呈する場合は、卵母細胞に囲まれた核小体 (SN) (図 1 a)、として分類します。</li>
	<li>彼らの核小体がヘキスト正クロマチンで囲まれていないとショーを分散させる (図 1 b) 核内多色スポットない囲まれた核小体 (NSN) として卵母細胞を分類します。</li>
</ol>4. ニューラル ネットワーク ベースの卵母細胞の分類
<ol><li>4 つの準備 5% 熱不活化牛胎児血清 2 mM L グルタミン 5 mM タウリン、100 U/mL ペニシリン、75 μ g/mL ストレプトマイシン、35 ミリメートルのガラスに 23.5 μ g/μ L ナトリウム ピルビン酸と α MEM 培 2 μ L 水滴シャーレの底し、でそれらをカバー予め温めておいた (37 ° C) および培地の蒸発を避けるためにあらかじめ釣合い鉱物油。 生きている細胞スクリーニング システムを使用している場合注意: 滴特定の距離をガイドラインとして 7 mm × 7 mm の描画グリッドを使用します。</li>
	<li>転送 3-4 卵にドロップします。</li>
	<li>コマ撮り録画ソフトウェアを搭載したライブ細胞スクリーニング システム ガラス底ペトリ皿に配置 (参照材料表)、安定した温度 (37 ° C) と CO2 環境で卵成熟過程の観測が可能濃度 (5%)。</li>
	<li>コマ撮り録画ソフトウェアを開きます。画面の左側にある卵子と右側にあるすべての設定ボタンのライブ ショット ウィンドウが表示されます。</li>
	<li>画面中央に位置する卵子のセンターを選択します。必要な場合は、フォーカスを調整します。</li>
	<li>Phのボタンを選択し、192; DIA ランプを設定露出時間 1/125 s;得るために1.99;1600 ×1200の解像度。</li>
	<li>FL2を選択し、ダイヤ ランプを5に設定露出時間 3 s;得るために2.37;1600 ×1200の解像度。</li>
	<li>文化ドロップ内卵子の位置を記録するために新しいポイントボタンを選択します。各卵母細胞文化ドロップ内の同じルーチンを繰り返します。</li>
	<li>時間を選択し、 8 分と合計時間 15 hまでに買収のサイクルを設定します。</li>
	<li>記録サイクルを開始するコマ撮りを開始を選択します。</li>
	<li>MATLAB ソフトウェアを開き、書き込み >>cell_piv;PIV ツールを選択します。</li>
	<li>記録映画が含まれているフォルダーを選択し、全体像をロードする .jpeg ファイルのいずれかを選択します。各卵母細胞領域ツールを選択をクリックして利益 (率 ROI) の領域を選択します。</li>
	<li>[ファイル] メニューでPIV プロセスを選択します。ビューアー ツールボタンを選択します。 注: プログラム ROI 内の速度ベクトルを計算し、.mat ファイルとして保存、データ ファイルが自動的に生成されます。</li>
	<li>.Mat ファイルを開くを選択します。ボタン選択 ROI ツールを選択し、卵母細胞の輪郭に沿って円を描きます。ファイル] メニューの [保存] をクリックします。 注: この地域でベクトルが表示されます、グラフ ウィンドウで振幅データ更新この新しい投資収益率。</li>
	<li>オープン全体時間経過フレームの収集した振幅データ記録と、フレーム シーケンスの行を列のスプレッドシートに各単一卵母細胞を分析します。</li>
	<li>10 サブグループに NSN と SN の卵母細胞のデータをまとめます。</li>
	<li>繰り返しフィード フォワード人工ニューラル ネットワークを訓練する 9 のサブグループを使用 (ファン) とブラインド テスト 1 サブグループ。<ol>
<li>別に繰り返し 9 回ブラインド テストのサンプル (10 倍クロス検証) を変更します。</li>
	</ol>
</li>
	<li>MATLAB を開き、カスタム スクリプトメインを実行し、リクエストでは、訓練データ (train.txt)、(通常から分析するトレーニングとインターバル撮影の間隔の使用 NSN と SN の卵子の数を持つファイルの名前を挿入フレーム フレーム # 112-113 に # 1-2)。</li>
	<li>トレーニングを実行する入力を押します。</li>
	<li>テスト データ (test.txt) を含むファイルの名前を挿入します。</li>
	<li>MATLAB のワークスペースでベクトルtest_outputs_cytを開きます。 注: 最初の行、それぞれ NSN と SN の卵子の本当の陽性 (TP または True NSN) および偽陽性 (FP または False NSN) を入手する確率を示す画面上にウィンドウが表示されます。代わりに、2 行目で、偽陰性 (FN または False SN) および NSN と SN の卵子の真のネガ (テネシー州または True SN) をそれぞれ取得する確率。</li>
	<li>数式を使用して: (TP+TN)/(TP+FP+FN+TN)、ファンの確度、パーセンテージを計算します。</li>
</ol>

<ol>
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著者が明らかに何もありません。

1 つはこのプロトコル マウス卵母細胞と他の種のそれらの実行中の世話をする必要がありますいくつかの重要な手順があります。コンパニオンの積雲からの分離細胞のトリガーに卵母細胞必要がありますすぐに記録の低下に転送一度彼らの卵胞から分離、GV の MII 遷移の開始。議定書への可能な変更が Coc 分離に使われる M2 媒体に 3-イソブチル-1-メチルキサンチン (IBMX) の付加であります。IBM...

女性の不妊は、女性数の増加に影響を与える検査です。世界保健機関によると約カップルの 20% が不妊、女性不妊原因の 40% です。さらに、がん治療を受ける女性の 3 分の 1 (300,000/年、米国やイタリアで 30,000/年それぞれ) 早期の卵巣の失敗を開発。
がん患者の不妊を防ぐため戦略は、MII ステージ (GV-MII 切り替え) に GV 卵子の体外成熟 (IVM) 続いて、がん治療前に卵胞の凍結と分離です。受精と発生過程と全体的な妊娠成功1,2卵子の発生能の非侵襲的なマーカーの可用性が向上します。
ヘキスト 33342、觀螢光色素で染色後に観察されたクロマチン構成に基づいて完全育てられた卵母細胞が囲まれた核小体 (SN) やない囲まれた核小体 (NSN)3のいずれかに分類されます。異質染色質組織以外にも卵母細胞のこれらの 2 つのタイプ表示多く形態的, 機能的違い3,4,5,6,7,8 ,9, 減数分裂と発達能力を含みます。体外成熟および卵巣から分離されたとき両方が MII 期卵子に達するの入力精子受精後 2 細胞期に開発、用語9SN クロマチン組織にのみ発展するかもしれない。有能と無能な卵母細胞を選択するための分類法として良い, 主な欠点は螢光色素自体と、上記のすべての検出に使用される紫外線が細胞に突然変異誘起効果は。
これらすべての理由から、我々 は同じ高い分類精度を維持しながらヘキストの使用を置き換えることができます SN または NSN のクロマチン構造に関連付けられている他の非侵襲的なマーカーの検索。細胞質の運動速度 (CMVs) の時間経過の観察は、細胞状態の特徴的な機能として現れています。たとえば、最近の研究は、CMVs の間の関連付けは、受精の時間と能力をマウスとヒトの受精卵の着床前の完全なと完全な長期的開発10,11で記録を示した。
これらの以前の研究に基づいて、ここで述べたい発達有能か無能なマウス完全育てられた卵5,6,7,8の認識のためのプラットフォーム。プラットフォームは、3 つの主な手順に基づいて: 1) から胞状卵胞由来卵子は最初囲まれて核小体 (SN) またはない囲まれた核小体 (NSN); としてクロマチン構成いずれかに基づいて分類されます各単一卵子の GV の MII の移行中に発生した CMVs の 2) コマ撮り画像が撮影され、粒子画像流速測定法 (PIV); と分析・ 3) PIV による解析で、フィード フォワード人工ニューラル ネットワーク (ファン) ブラインド分類12,13。私たちは、テストおよび他の哺乳類種 (例えば牛、猿、人間) ために使用する利用可能な準備ができているし、マウス用に設計されたプロシージャの最も重要な手順の詳細を与えます。

<table border="0" cellpadding="0" cellspacing="0" width="1257">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Folligon</td>
			<td style="width:239px;">Intervet</td>
			<td style="width:157px;">A201A02</td>
			<td style="width:499px;">Hormonal treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Hoechst 33342&#160;</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">B2261</td>
			<td>For oocyte heterochromatin staining</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:363px;">Cell culture Petri-dish 35 mm x 10 mm&#160;</td>
			<td style="width:239px;">Corning&#160;</td>
			<td style="width:157px;">430165</td>
			<td>For COCs isolation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">EmbryoMax M2 Medium (1X), Liquid, with phenol red</td>
			<td style="width:239px;">Merck-Millipore</td>
			<td style="width:157px;">MR-015-D</td>
			<td>For COCs isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Alpha medium (1X) + Glutamax&#160;</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">M4526</td>
			<td>For oocyte in vitro maturation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture Petri-dish 35 mm glass-bottom&#160;</td>
			<td style="width:239px;">WillCo&#160;</td>
			<td style="width:157px;">GWSt-3522</td>
			<td>For imaging experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">BioStation IM-LM&#160;</td>
			<td style="width:239px;">Nikon</td>
			<td style="width:157px;">MFA91001</td>
			<td>Live cell screening system&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Pasteur pipette</td>
			<td style="width:239px;">Delchimica Scientific Glassware</td>
			<td>6709230</td>
			<td>For follicles manipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mineral oil</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">M8410</td>
			<td>To prevent contamination and medium evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Penicillin / Streptomycin</td>
			<td style="width:239px;">Life Technologies</td>
			<td style="width:157px;">15070063</td>
			<td>To prevent medium contamination&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">ML16141079</td>
			<td>For making up &#945;MEM medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine&#160;</td>
			<td style="width:239px;">Life Technologies</td>
			<td style="width:157px;">25030</td>
			<td>For making up &#945;MEM medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">T0625</td>
			<td>For making up &#945;MEM medium&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">A3310</td>
			<td>For making up &#945;MEM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate&#160;</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:157px;">&#160;P4562</td>
			<td>For making up &#945;MEM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zoletil (Tiletamina and Zolazepan cloridrate)</td>
			<td style="width:239px;">Virbac Srl</td>
			<td style="width:157px;">QN01AX9</td>
			<td>For mice anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:363px;">Cell_PIV sofware&#160;</td>
			<td style="width:239px;">Kindly provided by Dr. Shane Windsor, University of Bristol, UK</td>
			<td style="width:157px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;-</td>
			<td style="width:499px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;-</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MATLAB</td>
			<td style="width:239px;">The MathWorks, Natick, MA&#160;</td>
			<td style="width:157px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;-</td>
			<td>For multi-paradigm numerical computing</td>
		</tr>
	</tbody>
</table>

a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes

不妊クリニック発達有能と無能な卵全体の妊娠の結果を向上させる非侵襲的な手順を使用してを選択する機能の恩恵。我々 は最近、卵核胞 (GV) から体外成熟細胞運動の分析に続いて、中期 II ステージ中にマウス卵母細胞の顕微鏡のライブ観察に基づく分類方法を開発この時間経過の期間中に発生しています。ここでは、このプロシージャの詳細なプロトコルを提案する.卵は、完全育てられたから胞状卵胞から隔離され、37 ° C、5% CO2で時間経過の分析のため、顕微鏡内 15 時間培養します。写真は、8 分間隔で撮影されます。各卵子の培養期間中に発生する細胞質の運動速度 (CMVs) のプロファイルを計算する粒子画像流速測定法 (PIV) メソッドを使用して画像を分析します。最後に、それぞれの単一の卵母細胞の CMVs は主務が発達 91.03% の精度で無能か配偶子の確率を予測する数理分類ツール (フィード フォワード ニューラル ネットワーク、ファン) を介して供給されます。このプロトコルは、マウスの設定は、人間を含む他の種の卵母細胞に今テストでした。

図 2は、それぞれ先頭 (GV) と IVM プロシージャの終わり (MII) 代表的な発達有能で無能な卵を示します。完全育てられたマウス卵母細胞の IVM 培養 15 時間中に発生します。時間経過の観察は減数分裂の進行を記録し、GVBD と第一極体の押し出しなどを含む、主要な減数分裂イベントを検出します。
分析?...

Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

ここでは、中期 II ステージに卵核胞から体外成熟中に実行される卵発達能力の非侵襲的評価するためのプロトコルを提案する.このメソッドは、粒子画像流速測定法 (PIV) ニューラル ネットワーク解析とタイムラプス イメージングを組み合わせます。

ニューラル ネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

a-neural-network-based-identification-developmentally-competent-or

Research

JoVE Journal

Developmental Biology

6.6K ビュー.  University of Pavia. ここでは、中期 II ステージに卵核胞から体外成熟中に実行される卵発達能力の非侵襲的評価するためのプロトコルを提案する.このメソッドは、粒子画像流速測定法 (PIV) ニューラル ネットワーク解析とタイムラプス イメージングを組み合わせます。

ビデオ: A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

無血清単層培養中のマウス胚性幹細胞の神経分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

発生生物学

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

早期着床後のマウス胚への細胞やDNAの正確​​ローカライズ転送する方法

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

核小体クロマチン組織に基づいてマウス洞卵母細胞の単離とキャラクタリゼーション

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

単離と大人の犬の海馬神経前駆体の拡大

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

フローサイトメトリーによるマウス胚における赤血球系前駆細胞およびその子孫の同定

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

卵母細胞のマイクロインジェクションによる遺伝子改変マウスの作製

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

マウスの早い妊娠の特性の三次元の高周波超音波の新規な使用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

神経クレスト細胞の形成と移動の研究のためのマウスからの原発性頭蓋神経クレスト細胞の解剖、培養および分析

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

マイクロCTイメージング用成人および早期産後マウス肺の血管鋳造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

未熟ネコの最小体積ガラス化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

ニューラル ネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定

ニューラルネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定