Name: dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment
Uploaded: 2018-01-13T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

この作品は、(社) にドイツ研究振興協会 SFB688 によって支えられました。

マウス繁殖は Regierung Unterfranken によって承認された、すべてのドイツ語、欧州連合の法律、実験動物の管理と使用に関する規制に従い、すべての解析を行った。
1. コーティングのマイクロタイター プレート α4β1 インテグリンとEx 子宮尿膜培養
<ol><li>200 μ g/mL 滅菌リン酸緩衝生理食塩水 (PBS) で凍結乾燥させたマウス α4β1 インテグリンを再構成します。予め温め...

<ol>
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プロの接着細胞外マトリックス分子フィブロネクチンなどの豊富な源である血清の存在下で尿膜外植体は容易に様々 なプラスチックやガラス、フィルター表面9,16にアタッチされます。したがって、実行される分析の目的は、遺伝子組み換えの効果を具体的に検討する場合、尿膜の添付ファイルに他の処理固定化 α4β1 は、非特異的結合部位 (例?...

胎盤が子宮環境の胚の成長と開発のために不可欠です。それは内分泌と免疫器官としての酸素代謝物、胎児と母体の循環とも機能間の栄養交換のためのインターフェイスを構成します。胎盤発育を損なう内因性または外因性の侮辱は、子宮内胎児発育遅延、胎児の損失、または妊娠合併症、マウスと人間1の両方につながることができます。対象となるマウスを異常な胎盤の生理を引き起こす突然変異の数が 219 遺伝子多型のマウスのゲノム情報学 (MGI) ウェブサイトに現在表示されている2を増やす続けます (http://www.informatics.jax.org/vocab/ mp_ontology/MP:0010038)、これらの表現型の多くはよく機械論的視点から理解されていません。に加えて遺伝子変異、小分子薬、モノクローナル抗体、毒素、病原体、余分な代謝物や様々 な環境要因や影響を与える胎盤開発関数3,4,5,6,7します。 まだ、簡単な生体外モデル胎盤の開発で重要な手順が不足している試金。
(臍帯発達前駆体) 尿膜が絨毛膜8に向かって育つ芽として胚の後部の端から現れるとき、初期の経産婦でマウス胎盤の発育が開始されます。尿の芽の外側の層の Chorioadhesive セルは、添付ファイルおよび絨毛膜 (漿「融合」) の表面に尿膜の広がりを仲介します。絨毛膜はその後に尿膜壁血管成長迷路、栄養素と酸素が密接に並置母体血と胎児血管9 間交換される胎盤内層を形成する絨毛に折る ,10。
絨毛膜へ尿の付着迷宮形成の初期および重要なステップであり、このプロセスの欠陥は midgestation1胚性致死性の最も一般的な原因のうち。マウス突然変異体の数が、漿添付ファイル10が発生するが失敗し、MGI データベース現在異常な漿融合 (http:// によって特徴付けられる 108 マウスの突然変異体の一覧を記載されてwww.informatics.jax.org/vocab/mp_ontology/MP:0002824)、血管細胞接着分子 1 (VCAM 1、尿中胚葉に発現) と (は絨毛の中皮に表される α4β1 インテグリンの細胞間相互作用初期胚胚体外胚葉由来) 漿添付ファイルと融合11,12,13のため不可欠であることが表示されます。(E) 7.5 日約 VCAM 1 が尿の茎の遠位 3 分の 2 以内単位があることを示している全体マウント野生型胚の免疫組織化学的染色13 (1-4 体節期)、およびその発現状態のまま高漿の添付ファイルと - 融合11,12。絨毛膜尿愛着の障害は、通常、子宮の芽の組織学的解析による検出します。ただし、尿膜サイズはの間と同じ発達段階14リットル内でも高い生理学的変数であり、尿は、物理的な接触をするのに十分な大きさに達したときに絨毛膜にのみ添付できます。この自然の変動を反映して、漿添付が行われる間のいつか 〜 E 8.0 と 9.0 E子宮内、および組織によって、このプロセスの統計的信頼性の高い評価は大きい数の解析に依存したがって淘汰適切な発達段階で十分な検体を採取します。
ここでは、尿の添付ファイル子宮 ex少ない尿膜のサイズに依存している評価方法について述べる。妊娠マウスの子宮からマウス胚とその allantoides の郭清を紹介 〜 8 日のポストの coitum 定め(dpc; dpc 0.5 が膣にプラグインの検出を示す)、固定化 α4β1 の外植体尿膜の後続の文化とインテグリンです。このメソッドは、添付ファイルおよび複数並列で、異なる連続した時点で尿膜外植体の挙動を広がりの急速な機能評価できます。プロトコルを使用することがあります尿膜添付ファイル前のヴィヴォに及ぼすターゲット変異、薬、または様々 な環境要因の画面に。

<table border="0" cellpadding="0" cellspacing="0" width="1607">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">C57BL/6J wildtype mice</td>
			<td style="width:256px;">The Jackson Laboratory</td>
			<td style="width:119px;">Stock No:&#160;000664</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 % (v/v) Ethanol</td>
			<td style="width:256px;">VWR International</td>
			<td>930031006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>11000-12</td>
			<td style="width:915px;">Forceps used to open the abdominal cavity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Micro dissecting forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>Dumont # 5 medical biology forcepts with fine, sharp tip.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fine scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>14094-11</td>
			<td>Straight blades.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>15012-12</td>
			<td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microspoon</td>
			<td style="width:256px;">Carl Roth</td>
			<td>AT18.1</td>
			<td>5 mm Spoon diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtiter plates</td>
			<td style="width:256px;">ibidi</td>
			<td style="width:119px;">81501</td>
			<td>We use uncoated, sterile angiogenesis slides (internal volume 50 &#181;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">3.5 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150288</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large orifice pipette tips&#160;</td>
			<td style="width:256px;">Biozym</td>
			<td style="width:119px;">VT0140X</td>
			<td>Low binding pipette tips, 200 &#181;L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a4b1 integrin</td>
			<td style="width:256px;">R&#38;D Systems</td>
			<td style="width:119px;">6054-A4</td>
			<td>Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>Without Ca2+ and Mg2+.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:319px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-03600</td>
			<td style="width:915px;">The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:319px;">Penicillin/Streptomycin</td>
			<td style="width:256px;">Gibco (ThermoFisher Scientific)</td>
			<td style="width:119px;">15140-122</td>
			<td>100 x (10,000 U/mL Penicillin and 10,000 &#181;g/mL streptomycin) stock solution.&#160;Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">L-Glutamine</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-80100</td>
			<td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fetal bovine serum&#160;</td>
			<td style="width:256px;">Biochrom</td>
			<td style="width:119px;">S 0115</td>
			<td>We use heat-inactivated FBS (heated to 56 &#176;C for 30 min with mixing to inactivate complement).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin (BSA)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">A7030</td>
			<td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &#181;m pore size and low protein-binding membrane, and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">para-Formaldehyde</td>
			<td style="width:256px;">Carl Roth</td>
			<td>0335.2</td>
			<td style="width:915px;">To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &#176;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &#176;C for approximately four weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Triton X-100</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">X100</td>
			<td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Normal goat serum</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>G9023</td>
			<td>To block non-specific binding of antibodies in immunofluorescence analyses.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a-CD31 Antibodies</td>
			<td style="width:256px;">BD Biosciences</td>
			<td>550274</td>
			<td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary goat anti-rat antibodies</td>
			<td style="width:256px;">Thermo Fisher</td>
			<td>A-11006</td>
			<td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mowiol 4-88</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>81381</td>
			<td>Mounting medium for cytochemistry. MW ~31,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Labovert&#160;</td>
			<td style="width:256px;">Leitz</td>
			<td style="width:119px;">Labovert&#160;</td>
			<td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">M80</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">M80</td>
			<td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DM4000B</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">DM4000B</td>
			<td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Digital camera</td>
			<td style="width:256px;">JVC</td>
			<td style="width:119px;">KY-F75U</td>
			<td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:319px;">Confocal microscope</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">SP5</td>
			<td style="width:915px;">Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td>
		</tr>
	</tbody>
</table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

胎盤は、成長と哺乳動物の胎児の開発のために不可欠です。このため、多数の遺伝子変異と胎盤の開発または機能を妨げる可能性が高いのも環境の侮辱が、マウスとヒトの妊娠初期の損失にあります。それにもかかわらず、画面の胎盤形成に対する潜在的な影響に簡単な生体外の試金が不足しています。ここでは、モデリングにおける胎盤絨毛膜に尿膜壁添付ファイルから成っている最初の重要なステップに焦点を当てます。Chorio 擬態の基板となる固定化 α4β1 インテグリンの尿外植片の添付ファイルを迅速に評価する方法について述べる。この体外アプローチにより、添付ファイルと異なる連続した時点で複数の尿膜外植体の挙動を拡散の定性的な評価です。プロトコルは、ターゲットを絞ったマウス突然変異、薬、または妊娠合併症や尿膜添付ファイル前のヴィヴォの胎児の損失にリンクされているさまざまな環境要因の効果を調べるために使えます。

このプロトコルを特定しマウス allantoides前のヴィヴォ、外植体について述べるし、体内漿融合中 α4β1 インテグリンに尿膜添付ファイル、重要なプロセスの定性的な評価の詳細。図 1 a-Hに示す代表的な結果は、妊娠子宮から始まって尿膜分離の連続した手順を示します。脂肪組織と血管 (図 1...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

モデル漿添付ファイル、胎盤形成の最初のステップの in vitroアッセイについて述べる。プロトコルは、固定化 α4β1 インテグリンのマウス allantoides の郭清と植の文化を示しています。尿膜の添付ファイルは、あらかじめ決められた時点で病理組織学的に評価されます。

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Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

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