The authors would like to thank Mark Bishop, Mathew Zverev, Victoria Marsh-Durban, Adam Blackwood and Sylvie Robine. This work was funded by a programme grant from Cancer Research UK.

<ol>
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The authors have nothing to disclose.

遺伝子および細胞の機能を分析するために、条件CRE-LOXトランスジェニックマウスを使用すると、一般的に使用されるアプローチです。これらのモデルは、特定し、特徴づける幹細胞2,4-6をし、病気25におけるその役割を理解するために腸で大きな成功を収めて使用されています。完全にこれらのモデルを活用するために、正しく解釈されるデータを可能にするために、システムの総合的な特性評価が必要です。これらのシステムの完全な理解が原因遺伝子はほとんど孤立細胞型または位置、生物学的知識及びCre発現を誘導するために使用されるシステムの非効率性の欠如に特定されないように達成することは困難です。ここで説明する方法は、我々は実験的なデザインと既存の知識のアプリケーションを介してこれらの問題を克服する方法を示します。我々は、ここで紹介するテクニックは一般的なものとムリを調査する任意の研究に利用することができる特定の研究の質問に答えるために、これらのメソッドを使用しましたがNE腸。 腸組織の作製 強固な結果を確保するための重要なステップは、厳密に付着適時および固定プロトコルで処理する必要がある組織の採取及び処理です。ほぼすべての重要な問題は、下流の組織乾燥アウトおよび/または不完全な固定に関連するアーティファクトに起因することができます。タイミングは、組織構造および/または核酸およびタンパク質の分解を防ぐために重要です。不完全または熱心固定は、組織化学的解像度の損失につながることができます。固定液の浸透がIHC分析の際に「潮マーク」として観察することができる腸陰窩内解像度の損失をもたらすことができるようにするにはあまりにも厚く、不十分な時間やセクションに不完全な固定。さらに核βカテニンはすぐにプロのない限り、核の外に拡散することができるように、それは、固定が長すぎるために拡張されないことが重要ですcessedとホルマリン固定次の埋め込みワックス。 ISCのニッチでパネート細胞の役割 ここに示されたデータは、効果的に、ISCの損失以下の成人の腸内で暗号再生におけるパネート細胞の重要性を示しています。しかしそこにAH-CREはVIL-のCre-ER T2をターゲットことのISCの人口を惜しみ可能性が残りました。天ら26はエレガント LGR5 ハイテクのISCがLGR5 LO予備のISCの人口に置き換えられていることを実証しました。今はこれらのISCが原因で分泌細胞前駆体6,7として同定された予備の集団にAH-CREシステムに免れている可能性が高いようです。 ISCの状態に戻すために必要なときに、これらの分泌細胞前駆体を支える成熟パネート細胞の重要性が答えられることを残ります。パネート細胞は、Iを構成するようにSCニッチ8とカロリー摂取量27と炎症28にISC応答の調節に役割を果たし、それは彼らの看護の機能は、独自の前駆体に拡張される可能性が残っています。 新しいアプローチとテクノロジモデルヒト結腸直腸癌を効果的に ISCの発見は、今やクラークら9によって概説腸生物学および疾患における遺伝子と細胞の役割を調べるための新しいマウスモデルを生成するために使用されている遺伝子の同定につながりました。この技術の唯一の制限は、Creタンパク質を発現する遺伝子の同定です。現在のISCは定期LGR5遺伝子発現パターンに基づいて、条件付きトランスジェニックマウスを用いて調べました。 LGR5プロモーターからのCreを発現するマウスは 、APC、最も一般的に大腸癌で変異遺伝子（CRCを削除するために使用されています）、起源25のセルとして、ISCを実証します。選択的に削除するこれらの細胞内の他のCRC遺伝子は、疾患の進行への洞察を提供し、広がっているなど。PTEN 29。 ISC機能へのさらなる洞察は、ヒトジフテリア毒素受容体（DTR）遺伝子を用いて、マウスの細胞を発現性LGR5を切除することにより取得されるLGR5座 26にノックしました。他の戦略は、変異タンパク質30の継続的な可逆的発現を可能にテト-Oシステムを使用しています。異なるセル31との位置32,33における遺伝子（複数可）を変更するためにこれらのツールを使用すると、癌は、進行を開始し、34の転移方法を理解するために使用されます。あるいは、眠れる森の美女トランスポゾンシステムを用いて、突然変異誘発は、CRCの新しいドライバを識別しています。マウス、技術や遺伝子変化戦略の継続的な開発は、より多くの患者関連するモデルを開発し続けています。 新しいメートルethodsは、腸管上皮とのISCの特徴付けのために開発されてきました。上皮細胞型の割合の特徴付けは、レクチンおよびCD24 35の示差的な発現に基づいてフローサイトメトリーを用いて達成することができます。潜在ISC生物学および疾患におけるそれらの役割を理解する上で最大の進歩は 、ex vivoオルガノイド培養系36を用いて説明します。このシステムは、彼らが複製し、より生理学的に関連する方法で差別化、3D、培養に正常および悪性のISCを可能にします。これらは個別化医療37への道を切り開いて、in vitroで患者サンプルに対する薬物の直接的な試験を可能にすることが期待されています。

哺乳動物の腸の管腔表面は、突起のような陰窩と指の繰り返し単位を備え、内腔に突出する絨毛は、と呼ばれます。 4日目1 -この表面は、ほぼ毎3完全な自己再生を受ける上皮の連続シートです。最初LGR5遺伝子 2,3の発現によって同定した。この動的な組織が ​​急速にサイクリング幹細胞（も陰窩ベース円柱細胞として知られているのISC）の人口でサポートされています。これらの細胞は、リーベルの陰窩の下部に特化したニッチに存在します。当初、のISCが急速にサイクリングた発見は、幹細胞が本質的に静止したことを支配する考えと耳障りでした。それは静止ラベルの人口は、陰窩のベースに対して4位置に細胞を保持すると仮定されたLGR5 + ISCの同定前、のISC 1でした 。最近の研究の時間今主に運命隣国4,5によって規制されている各陰窩における等効力サイクリングのISCのプールがあることを実証することによって、これらの観察を両立。イベントでは、彼らは、これらは通常、分泌系統にコミットしているが、ISCの人口は6を損傷した場合のISCに戻すことができ、静止細胞に置き換えることができます失われます。 ISCの隣人はのISCまたはその娘細胞のいずれかとすることができます。 ISCは、増殖しているライン腸管腔1上皮シートを備える特殊な細胞型に分化ナイーブ娘細胞を産生します。ゴブレット、腸、腸、房およびM細胞は、様々な吸収や調節機能を提供腔表面に上向きに移動するが、パネート細胞は、それらがのISCと混ざり存在陰窩の底に残っています。近年では、ナイーブdaughtの割合ことが実証されています分泌系統宛てER細胞が傷害6,7時に、ISCに戻すことのできるLGR5 LO細胞を保持静止ラベルです。 原因陰窩再生におけるその重要性に優先順位をのISCとその近隣諸国、特にパネート細胞間の相互作用を理解する上に置きました。パネート細胞はのISC 8をサポートしてニッチで重要な役割を果たしています。殺菌製品に加えて、パネート細胞は、ISCの再生または分化を支配する経路を活性化するシグナル伝達分子を生成します。以前の研究は、彼らが自分の娘パネート細胞8が提供する本質的なニッチ信号を競うことができたときにLGR5 +のISCにのみ存在することを示しました。これらの研究は、通常のLGR5 +のISCではなく、それらが損傷し、LGR5 LO集団からの補充を必要としている状況でパネート細胞の役割を調べました。 <pクラス= &quot;jove_contentは&quot;&gt;我々はトランスジェニックマウスモデル9,10を用いて細胞および/ ​​または遺伝子の機能的役割を調べ腸生物学とモデル疾患を理解するために。頻繁にこれらのモデルは、条件付き遺伝子（複数可）9,10を変更するには、CRE-LOX 技術を活用しています。のCre（組み換えを起こす）リコンビナーゼは、バクテリオファージP1から分離され、インテグラーゼファミリーの部位特異的リコンビナーゼである。Creを&#39;は定義された34塩基対の間の部位特異的組換えを触媒しますロックスP &#39;（クロスオーバーP1の軌跡χ）サイト。マウスは遺伝的にCreリコンビナーゼの発現の際に切除される関心領域に隣接するLoxP部位を含むように操作されています。セルまたは発生特異的プロモーターにCre遺伝子の発現をリンクすると、変更は、空間ファッション9,10で行われるために、これは胚性致死突然変異を克服するのに特に有用であることができます。さらに受容体経路にCre発現を結びます、それは一時的な変更を可能にし、人為的に活性化させることができます。 この技術を用いて、我々は、腸上皮にCatnB遺伝子 11を不活性化。 βカテニン、CatnB遺伝子産物は 、ISCのホメオスタシスを支配する標準的なWntシグナル伝達経路の重要な調節因子です。この戦略を使用して、前の2つの研究では、矛盾する結果12,13を生産しました。 Fevr ら12の研究は、幹細胞および腸の恒常性の喪失を示しました。アイルランドらに対し、14の研究では、細胞生存率の低下を以下陰窩-絨毛軸がCatnBを発現する野生型細胞からの再増殖したことを報告しました。これらの研究における主な違いは、腸の上皮でのCreを発現するために使用されるプロモーターでした。 Fevr らの研究は、Tを投与することによって活性化することができる、エストロゲン受容体に結合されたビリン遺伝子プロモーターを使用しましたamoxifen（VIL-のCre-ER T2の ）15,16。対照的にアイルランドらは、生体異物βナフトフラボン（AH-CRE）に応答して、Cre発現を駆動するために、ラットチトクロームP450A1（CYP1A1）遺伝子のプロモーター要素を利用します。これら異なるシステムの特徴は、これらの異なった観察を考慮するために、2つの仮説を生成しました。 CatnBをより効率良くサブ再増殖レベルへのISCの数を減らす、AH-CREに比べVIL-のCre-ER T2のシステムを使用して ISCで削除されていることを初めて。あるいは、それは、分化した細胞集団でCatnBの削除を差によるましました。 VIL-のCre-ER T2システムの対象AH-CREシステムに対し、陰窩および絨毛のすべての上皮細胞にのみ、ISCのニッチと陰窩の非パネート細胞を標的とします。これらのシステムはbehavを調べるための理想的なツールを提供しますISCのIORとパネート細胞との相互作用。ここでは、我々はパネート細胞は傷害17に腸の応答を媒介するのに重要な役割を果たしていることを決定するためにこれらのシステムを使用する方法に基づいていくつかの詳細なプロトコルを提示します。

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Acetate buffer</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 4.8ml 0.2M Acetic acid, 45.2ml 0.2M Sodium acetate &#38; 50ml distilled water</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>C/0400/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride</td>
			<td>Sigma</td>
			<td>A6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride solution</td>
			<td>*</td>
			<td>*</td>
			<td>2 M Acetic anhydride in 0.1 M triethanolamine hydrochloride</td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Sigma</td>
			<td>A5268</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue ph2.5</td>
			<td>*</td>
			<td>*</td>
			<td>To make 500ml: 15ml acetic acid, 5g Alcian Blue &#38; 485ml distilled water</td>
		</tr>
		<tr>
			<td>anti-digoxigenin alkaline phosphatase conjugated antibody</td>
			<td>Abcam</td>
			<td>ab119345</td>
			<td></td>
		</tr>
		<tr>
			<td>B(beta)-Naphthoflavone</td>
			<td>Sigma</td>
			<td>N3633</td>
			<td>BNF, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &#8211; store at -20&#176;C between uses, do not reheat more than twice.</td>
		</tr>
		<tr>
			<td>Bloxall</td>
			<td>Vector Labs</td>
			<td>SP-6000</td>
			<td></td>
		</tr>
		<tr>
			<td>BM purple</td>
			<td>Roche</td>
			<td>11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A4503</td>
			<td>Bovine serum albumin</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C/4920/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate Buffer/Antigen Unmasking Solution</td>
			<td>Vector Labs</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma</td>
			<td>C8627</td>
			<td></td>
		</tr>
		<tr>
			<td>Demucifiying solution</td>
			<td>*</td>
			<td>*</td>
			<td>For 500ml: 50ml glycerol, 50ml Tris 0.1M pH8.8, 100ml EtOH, 300ml saline (0.9% NaCl in water), DTT 1.7g.&#160; Demucifying solution can be made in advance and stored, but DTT sholud be added just before incubation (340mg/100ml).</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Life Technologies</td>
			<td>750023</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>101509944</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>O3690</td>
			<td>0.5M</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>E/0650DF/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>3000917</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin&#160;</td>
			<td>Sigma</td>
			<td>SLBL11382V</td>
			<td>Neutral buffered formalin</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F5786</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehye</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin</td>
			<td>Raymond A Lamb</td>
			<td>12698616</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>HBSS (-MgCl2+; -CaCl2)</td>
		</tr>
		<tr>
			<td>Hybridisation buffer</td>
			<td>*</td>
			<td>*</td>
			<td>&#160;5&#215; SSC, 50% formamide, 5% SDS, 1 mg/ml heparin, 1 mg/ml calf liver tRNA</td>
		</tr>
		<tr>
			<td>Hydroquinone</td>
			<td>Sigma</td>
			<td>H9003</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPACT DAB Peroxidase</td>
			<td>Vector Labs</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpress HRP Anti-Mouse IgG Kit</td>
			<td>Vector Labs</td>
			<td>MP-7402</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpress HRP Anti-Rabbit IgG Kit</td>
			<td>Vector Labs</td>
			<td>MP-7401</td>
			<td></td>
		</tr>
		<tr>
			<td>Intestinal tissue powder</td>
			<td>*</td>
			<td>*</td>
			<td>The small intestines of 5 adult mice were combined and homogenised in the minimum volume of ice cold PBS. 4 volumes of ice cold acetone were added to the homogenised intestine, which was mixed thoroughly and incubated on ice for 30 minutes. This was centrifuged and the pellet was washed using ice cold acetone. This was further centrifuged and the resulting pellet spread onto filter paper and allowed to dry. Once thoroughly dry the material was ground to a fine powder using a pestle and mortar.&#160;</td>
		</tr>
		<tr>
			<td>K-ferricyanide</td>
			<td>Sigma</td>
			<td>P-3667</td>
			<td></td>
		</tr>
		<tr>
			<td>K-ferrocyanide</td>
			<td>Sigma</td>
			<td>P3289</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Sigma</td>
			<td>L0380000</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacarn</td>
			<td>*</td>
			<td>*</td>
			<td>60% Methanol:30% Chloroform:10% Acetic acid</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>M/4000/17</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Labs</td>
			<td>S-1012</td>
			<td>NGS</td>
		</tr>
		<tr>
			<td>Normal rabbit serum</td>
			<td>Dako</td>
			<td>X0902</td>
			<td>NRS</td>
		</tr>
		<tr>
			<td>NTMT</td>
			<td>*</td>
			<td>*</td>
			<td>100 mM NaCl, 100 mM Tris HCl, 50 mM MgCl2, 0.1% Tween20, 2 mM Levamisole</td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Vector</td>
			<td>H-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBT</td>
			<td>*</td>
			<td>*</td>
			<td>0.5M NaCl, 10mM TrisHCL pH7.5, 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100x solutiuon.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (10x)</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>Dilluted 1:10 with distilled water to make 1x</td>
		</tr>
		<tr>
			<td>PLL slides</td>
			<td>Sigma</td>
			<td>P0425-72EA</td>
			<td>Poly-L-lysine microscope slides</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase k solution</td>
			<td>*</td>
			<td>*</td>
			<td>Dilute Proteinase K at 200 &#181;g/ml in 50 mM Tris, 5 mM EDTA.</td>
		</tr>
		<tr>
			<td>Ralwax</td>
			<td>BDH</td>
			<td>36154 7N</td>
			<td></td>
		</tr>
		<tr>
			<td>Reducer Solution</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 1g Hydroquinone, 5g sodium sulphite &#38; 100ml distilled water</td>
		</tr>
		<tr>
			<td>RnaseA</td>
			<td>Sigma</td>
			<td>R6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>*</td>
			<td>*</td>
			<td>0.9% NaCl in distilled water</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>I3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma</td>
			<td>S/1240/46</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver solution</td>
			<td>*</td>
			<td>*</td>
			<td>To make 100ml: 10ml Acetate buffer, 87ml distilled water, 3ml 1% silver nitrate</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>S/2120/53</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S6753</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Sigma</td>
			<td>239321</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>Sigma</td>
			<td>93017</td>
			<td>20x saline sodium citrate</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>Fisher Scientific</td>
			<td>12960495</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>TAM, inject without allowing the solution to cool too much as compound will drop out of solution. Solution can be re-used &#8211; store at -20&#176;C between uses, do not reheat more than twice.</td>
		</tr>
		<tr>
			<td>TBS/T</td>
			<td>Cell Signalling</td>
			<td>#9997</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine hydrochloride</td>
			<td>Sigma</td>
			<td>T1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma</td>
			<td>TP9416</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaMount</td>
			<td>Vector Labs</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield Hardset mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Labs</td>
			<td>PK-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Promega</td>
			<td>V3941</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal fixative</td>
			<td>*</td>
			<td>*</td>
			<td>2% formaldehyde, 0.1% glutaraldehyde in 1xPBS</td>
		</tr>
		<tr>
			<td>X-gal stain</td>
			<td>*</td>
			<td>*</td>
			<td>X-gal stain; 200ul X-gal (A) in 50ml solution B (0.214g MgCl2, 0.48g K-ferricyanide, 0.734g K-ferrocyanide in 500ml PBS).&#160; Solution B can be made up oin advance and stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X/0200/21</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocols for analyzing the role of paneth cells in regenerating the murine intestine using conditional cre-lox mouse models

7日 - 哺乳類の腸の上皮表面は、すべて3を更新動的な組織です。この更新プロセスを理解することは、急速にLGR5遺伝子の発現によって特徴付けられる腸管幹細胞（のISC）をサイクリングの人口を同定しました。これらは、損傷の際にそれらを置き換えることができるBMI-1発現によってマーク静止幹細胞集団、によって支持されています。これらの集団間の相互作用を調査すると、疾患や癌における役割を理解するために不可欠です。 ISCは、腸の表面に陰窩内に存在し、これらのニッチは、上皮を補充するには、ISCをサポートしています。それは以前にLGR5 ISCの&#39;&#39;幹細胞性」が「密接に隣接パネート細胞の存在に結びついていることが実証されているように、活性と静止のISCとの間の相互作用はおそらく、ニッチ内の他の分化細胞を含んでいます。条件CRE-LOXのマウスを使用モデル我々は、パネート細胞の存在下または非存在下で活性のISCの大部分を削除する効果を試験しました。ここでは、腸を特徴付け、パネート細胞はかなりの損傷後の回復を助けるのに、ISCのニッチ内の重要な役割を果たしていることを証明するために行わ技術と分析について説明します。

Watch this Scientific Journal Video about Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models at JoVE.com

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

腸の上皮幹細胞（のISC）は、パネート細胞と混在しています。これらの細胞は、のISCをサポートするISCの子孫を分化し、抗菌保護を提供しています。ここでは、我々はパネート細胞は腸管上皮を維持する上で重要な役割を果たしていることを確立するために、トランスジェニック条件付きマウスモデルを使用する方法を示します。

条件を使用して、マウス腸の再生成にパネート細胞の役割を分析するためのプロトコル<em&gt;のCre-LOX</em&gt;マウスモデル

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

protocols-for-analyzing-role-paneth-cells-regenerating-murine

Research

JoVE Journal

Developmental Biology

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

17.5K ビュー.  Cardiff University. 腸の上皮幹細胞（のISC）は、パネート細胞と混在しています。これらの細胞は、のISCをサポートするISCの子孫を分化し、抗菌保護を提供しています。ここでは、我々はパネート細胞は腸管上皮を維持する上で重要な役割を果たしていることを確立するために、トランスジェニック条件付きマウスモデルを使用する方法を示します。 

ビデオ: Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

トランスポゾンのシステムを用いたマウスの羊水細胞から多能性幹細胞の作製

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

発生生物学

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模擬微小重力下でのマウス前胞状卵胞の成長

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

マウス二次口蓋融合のライブイメージング

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL による破骨細胞文化アッセイのマウス骨髄破骨細胞形成における mTORC1 の役割を調査するには

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

線虫のニューロンに生殖細胞を再プログラムする RNAi と熱衝撃による転写因子の発現のアプリケーション

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

腸の生きている線虫のライソゾーム アルカリ化を評価します。

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

核の分布、蛋白質および細胞骨格を研究する線虫生殖細胞の解析

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

ノードと走査型電子顕微鏡と蛍光抗体法マウント全体を使用して Gastrulating のマウス胚における脊索板を可視化

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

骨組織の再生にプレドニゾロンの効果を研究する大人のゼブラフィッシュ傷害モデル

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

紫外C(UV-C)損傷に対する主なマウス眼表面細胞/幹細胞における酸化的損傷の評価

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...