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.

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The authors have nothing to disclose.

유전자와 세포의 기능을 해부 조건부 CRE-LOX 트랜스 제닉 마우스를 사용하는 것은 일반적으로 사용되는 접근 방법이다. 이 모델의 특성을 식별하고 줄기 세포를 2,4-6 질병 25에서의 역할을 이해하기 위해 소장에서 큰 성공으로 사용되어왔다. 이러한 모델을 충분히 활용하기 위해서는 올바르게 해석 할 데이터를 사용하는 시스템의 종합적인 특성을 요구한다. 이러한 시스템의 완전한 이해 인해 유전자 거의 독방 세포 유형 또는 위치, 생물학적 기술 및 Cre 호텔 발현을 유도하기 위해 사용되는 시스템의 비 효율성의 부족 특이성 없음으로 달성하기 어렵다. 여기에 설명 된 방법은 우리가 실험 설계 및 기존 지식의 응용 프로그램을 통해 이러한 문제를 극복하는 방법을 보여줍니다. 우리는 여기에 제시된 일반적인하고 Muri의 조사를 어떤 연구에 이용 될 수있는 기술 특정 연구 질문에 대답하기 위해 이러한 방법을 사용하지만NE 소장. 창자 조직의 준비 견고한 결과를 보장하기위한 중요한 단계는 엄격하게 부착 및 고정 적시 프로토콜에서 처리 될 필요가 조직의 수확 처리이다. 거의 모든 중요한 문제는 하류 티슈 건조 아웃 및 / 또는 불완전한 정착과 관련된 인공물에 기인 할 수있다. 타이밍은 조직 구조 및 / 또는 핵산 및 단백질의 분해를 방지하기 위해 매우 중요하다. 불완전하거나 지나치게 열성적인 고정은 조직 화학적 해상도의 손실이 발생할 수 있습니다. 시간이 부족하거나 정착 침투 IHC 분석에 따라 &quot;조류 마크&quot;로 관찰 할 수있는 장 지하실 내에서 해상도의 손실이 발생할 수 있도록 너무 두꺼운 부분으로 인해 불완전한 고정. 또한 그것은 핵 β-catenin이 바로 프로 않는 한 핵 밖으로 확산 될 수로 고정이 너무 오래 연장하지 않는 것이 중요합니다가공품의 수출입을 담 당하고과 포르말린 고정 다음 포함 된 왁스. ISC 틈새에서 Paneth 세포의 역할 여기에 제시된 데이터는 효과적으로 감소 ISC 다음 성인 부위에 토굴 재생성 Paneth 세포의 중요성을 보여준다. 그러나이 아 - CRE가 VIL-Cre 호텔 - 응급실 T2 대상 ISCS의 인구 여분 가능성을 유지했다. 티안 등. (26)은 우아 Lgr5 안녕하세요 ISCS는 Lgr5 싸다 예약 ISCS의 인구에 의해 대체되는 것을 보여 주었다. 지금이 ISCS 인해 예비 인구가 분비 세포 전구체 6,7로 확인 된에 AH-CRE 시스템에서 절약되는 것으로 보인다. ISC 상태로 복귀하는 데 필요한 때이 분비 세포 전구체를 지원하는 성숙한 Paneth 세포의 중요성은 대답 일이다. Paneth 세포를 구성하는 I로서SC 틈새 8 그들의 간호 기능을 자신의 전구체로 확장 할 가능성이 남아 칼로리 섭취량 (27)과 염증 28 ISC 반응을 조절하는 역할을한다. 새로운 접근 방식 및 기술을 효과적으로 모델 인간의 대장 암에 ISC의 발견은 지금 클라크 등의 검토에 의해 알 9 장 질환 및 생물학, 유전자 및 세포의 역할을 조사하는 새로운 마우스 모델을 생성하는 데 사용되는 유전자의 동정되었다. 이 기술에 대한 유일한 제한은 Cre 호텔 단백질을 발현하는 유전자를 확인한다. 현재 ISCS는 정기적으로 Lgr5 유전자 발현 패턴을 기반으로 조건부 형질 전환 마우스를 사용하여 조사하고 있습니다. Lgr5 프로모터로부터 발현 생쥐를 Cre 호텔은 APC, 가장 일반적으로 대장 암에서 돌연변이 된 유전자 (CRC를 제거하는데 사용되어왔다), 원점 (25)의 셀로서 ISC 시연. 선택적으로 삭제 이들 세포에서 다른 CRC 유전자는 질병의 진행에 대한 통찰력을 제공하고 확산된다 예. PTEN (29). ISC 기능에 추가 통찰력은 특히 절제 Lgr5 인간 디프테리아 독소 수용체를 사용하여 마우스의 세포를 발현에 의해 검색되고 (DTR) 유전자 Lgr5에 궤적 (26) 혔어요. 다른 전략이 돌연변이 단백질 (30)의 지속적인 가역 식을 수있는 구정-O 시스템을 사용합니다. 다른 셀 (31) 및 위치 (32, 33)의 유전자 (들)을 수정할 이러한 도구를 사용하는 것은 암이 시작하는 방법을 이해, 진보와 34 전이하는 데 사용됩니다. 또한 잠자는 아름다움 트랜스포존 시스템을 사용하여 돌연변이는 CRC의 새 드라이버를 식별한다. 마우스, 기술과 유전자 변형 전략의 지속적인 발전은 더 많은 환자 관련 모델 개발을 계속하고있다. 새로운 Methods는 장 상피와 ISCS의 특성을 위해 개발되었다. 유량을 이용하여 달성 될 수 상피 세포 유형의 비 특성화 계측법 렉틴과 CD24 (35)의 발현 차이에 기초. 잠재적으로 가장 큰 이해 ISC 생물학의 발전과 질병에서의 역할은 생체 organoid 문화 시스템 (36)을 사용하여 만들 수 있습니다. 이 시스템은 복제하고보다 생리 학적으로 중요한 방법으로 차별화 3D, 문화에 정상 및 악성 ISCS 수 있습니다. 이들 맞춤 의학 (37)에 대한 방법을 포장, 체외에서 환자 샘플에 약물을 직접 테스트를 가능하게 될 것으로 기대된다.

포유 동물의 창자 내강 표면이 돌기처럼 지하실과 손가락의 반복 단위 기능, 루멘 내로 돌출 융모를 불린다. 4 일 (1) -이 표면은 대략 매 3 완전한 자기 갱신을 거쳐 상피의 연속 시트입니다. 이 역동적 인 조직을 빠른 속도로 줄기 세포 순환의 인구에 의해 지원됩니다 (ISCS을, 또한 토굴베이스 원주 세포라고도 함) 처음 Lgr5 유전자 2,3의 발현에 의해 확인되었다. 이러한 세포 Lieberkuhn의 지하실 하단에 특화된 틈새에 존재한다. 처음 ISCS 빠르게 순환 하였다 발견 줄기 세포가 본질적으로 정지했다 지배적 아이디어와 불일치이다. 이 대기 라벨의 인구가 지하실의 기본에 대해 네 위치에서 세포를 유지하는 것으로 가정 된 Lgr5 + ISC의 식별에 이전, ISCS 1이었다. 최근 연구 시간지금처럼 주로 운명 이웃 4,5에 의해 조절되는 각각의 토굴에 효능이 동등 자전거 ISCS의 풀 (pool)이 있음을 보여줌으로써 이러한 관찰을 조정. 이벤트에서 그들은 이러한 일반적으로 분비 혈통에 최선을 다하고 있습니다 만 ISC 인구 6 손상된 경우 ISCS으로 되돌릴 수 있습니다 정지 세포에 의해 대체 될 수 있습니다 손실됩니다. ISC의 이웃 ISCS 또는 딸 세포가 될 수 있습니다. ISCS는 번성하는 선 장 루멘 (1) 상피 시트를 구성하는 특수 세포 유형으로 분화 순진한 딸 세포를 생성합니다. 잔, 장 내분비, 장 세포, 터프 트와 M 세포들이 다양한 흡 규제 기능을 제공 내강 표면을 위쪽으로 이동하지만, Paneth 세포들이 ISCS 섞여 존재 토굴의 바닥에 남아있다. 최근 몇 년 동안이 입증되었다 그 순진한 daught의 비율분비 혈통 향하는 어 세포는 손상 6,7에 ISC에 복귀 할 수있는 Lgr5 싸다 세포를 유지하는 대기 라벨입니다. 토굴에 의한 재생의 중요성에 순위가 ISCS과 이웃 특히 Paneth 세포 간의 상호 작용을 이해 넣었다. Paneth 세포 ISCS 8을 지원 틈새에 중요한 역할을한다. 살균 제품 이외에도 Paneth 세포는 ISC 갱신 또는 차별화를 지배하는 경로를 활성화 분자 신호를 생성한다. 이전의 연구들은 자신의 딸 Paneth 세포 (8)에 의해 제공되는 필수 틈새 신호를 경쟁 할 수있을 때 Lgr5 + ISCS는 존재할 수 있음을 보여 주었다. 이러한 연구는 정상 Lgr5 + ISCS과하지들이 손상 Lgr5 LO 인구에서의 보충을 필요로되는 상황에서 Paneth 세포의 역할을 조사 하였다. <p클래스 = &quot;jove_content은&quot;&gt; 우리는 형질 전환 마우스 모델 (9, 10)를 사용하여 세포 및 / 또는 유전자의 기능적 역할을 조사 장 생물학 및 모델 질병을 이해합니다. 자주 이러한 모델은 조건부 유전자 (들) 9,10을 수정 CRE-LOX 기술을 활용. Cre 호텔은 재조합 박테리오파지 (P1)에서 고립 된 인테그라 가족의 사이트 특이 적 재조합 효소이다 (재조합이되게합니다). Cre 호텔은 정의 (34) BP 사이에 위치 특이 적 재조합을 촉매로 &#39; 훈제 연어 P &#39;사이트 (크로스 오버 (P1)의 궤적 χ). 마우스는 유전자 Cre 호텔 재조합 효소의 발현에 절제되어 관심 지역의 측면에 loxP 사이트를 포함하도록 설계되었습니다. 세포 발달 또는 특정 프로모터 Cre 호텔 유전자의 발현을 연결하면 변경이 공간적 패션 9,10 이루어져야하는 경우,이 배아 치사 돌연변이를 극복하는데 특히 유용 할 수 있습니다. 또한 수용체 경로에 Cre 호텔 식을 연결하는,즉 인위적으로 활성화 될 수있는, 시간 변경을 허용한다. 이 기술을 이용하여, 우리는 장 상피에서 11 CatnB 유전자를 불 활성화. β 카테닌, CatnB 유전자 산물은 ISC 항상성에 적용되는 표준이 Wnt 신호 전달 경로의 조절 키이다. 이 전략을 사용하여 두 개의 이전 연구는 충돌하는 결과 12, 13을 생산했다. Fevr 등. (12)에 의한 연구는 줄기 세포와 장내 항상성의 손실을 보여 주었다. 아일랜드 외. 반면 14 연구 세포 생존율의 감소를 수행하는 것은 토굴-융모 축 CatnB 야생형 발현 세포에서의 증식을보고 하였다. 이 연구의 주요 차이점은 장내 상피 세포에 Cre 호텔을 표현하는 데 사용되는 발기인이었다. Fevr 등 알. 연구는 t를 투여함으로써 활성화 될 수있는 에스트로겐 수용체에 결합 villin 유전자 프로모터를 사용amoxifen (VIL-Cre 호텔 - 응급실 T2) (15, 16). 반면 아일랜드 등., 생체이 β-naphthoflavone (AH-CRE)에 대한 응답으로 Cre 호텔 식을 구동하기 위해 쥐 시토크롬 P450A1 (CYP1A1) 유전자의 프로모터 요소를 이용했다. 이러한 서로 다른 시스템의 특성은 서로 다른 관찰을 고려하여 두 가지 가설을 생성. CatnB보다 효율적하여 하위 레벨로 다시 채우기 ISCS의 수를 줄여, AH-CRE 비교 VIL-Cre 호텔 T2-ER의 시스템을 이용하여 ISC에서 삭제되는 제. 다른 방법으로는 분화 된 세포 인구에 CatnB 삭제 차동 예정이었다. VIL-Cre 호텔-ER T2 시스템 대상 AH-CRE 시스템 반면 crypt와 융모의 모든 상피 세포는 ISC 틈새과 토굴의 비 Paneth 세포를 대상으로합니다. 이러한 시스템은 검사 behav 이상적인 툴을 구비ISCS의 IOR과 Paneth 세포와의 상호 작용. 여기에서 우리는 우리가 Paneth 세포가 손상 17 장 반응을 중재에 중요한 역할을하는지 결정하기 위해이 시스템을 사용하는 방법에 따라 여러 가지 세부적인 프로토콜을 제시한다.

<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

칠일 - 포유류의 소장의 상피 표면은 매 3를 갱신 역동적 인 조직이다. 빠르게 Lgr5 유전자의 발현을 특징으로 장 줄기 세포 (ISCS)를 순환의 인구는이 갱신 프로세스를 발견 이해. 이러한 손상의 경우에 이들을 대체 할 수있는 BMI-1의 발현에 의해 표시 정지 줄기 세포 집단, 지원된다. 이러한 집단 사이의 상호 작용을 조사하는 질환과 암에 자신의 역할을 이해하는 데 매우 중요하다. ISCS는 장 표면에 지하실 내에 존재하는,이 틈새는 상피 세포를 보충에 ISC를 지원합니다. 것은 이미 Lgr5 ISC의 &#39;가 stemness&#39;밀접 그들의 이웃 Paneth 세포의 존재에 묶여 있다는 입증 된 바와 같이 활성 및 대기 ISCS 사이의 상호 작용 가능성, 틈새 내의 다른 분화 된 세포를 포함한다. 조건부 CRE-LOX 마우스를 사용하여모델 우리 Paneth 세포의 존재 또는 부재하에 활성 ISCS의 대부분을 삭제하는 효과를 시험 하였다. 여기에서 우리는 소장의 특성을하고 수행 기술과 분석은 Paneth 세포가 상당한 모욕 다음과 같은 복구를 돕기에 ISC 틈새 내에서 중요한 역할을한다는 것을 증명에 대해 설명합니다.

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

장 상피 줄기 세포 (ISCS)는 Paneth 셀과 혼합된다. 이러한 세포 ISCS을 지원 항균 보호를 제공 ISC의 자손을 차별화한다. 여기에서 우리는 우리가 Paneth 세포가 장 상피 세포를 유지하는데 중요한 역할을한다는 설정하는 형질 전환 조건부 마우스 모델을 사용하는 방법을 보여줍니다.

조건을 사용하여 쥐 소장에서 재생성 Paneth 세포의 역할을 분석하기위한 프로토콜<em&gt; Cre 호텔-LOX</em&gt; 마우스 모델

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

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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 조회수.  Cardiff University.  장 상피 줄기 세포 (ISCS)는 Paneth 셀과 혼합된다. 이러한 세포 ISCS을 지원 항균 보호를 제공 ISC의 자손을 차별화한다. 여기에서 우리는 우리가 Paneth 세포가 장 상피 세포를 유지하는데 중요한 역할을한다는 설정하는 형질 전환 조건부 마우스 모델을 사용하는 방법을 보여줍니다. 

비디오: 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.

트랜스포존 시스템을 사용하여 마우스 양수 세포로부터 다 능성 줄기 세포의 제조

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

연구

발생학

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.

생체 외에서 마우스 시뮬레이션된 Microgravity에서 Preantral 모 낭의 성장

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.

Mouse Secondary Palate Fusion의 라이브 이미징

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 기반 Osteoclast 문화 분석 결과의 마우스 골 수 mTORC1 Osteoclast 형성에의 역할을 조사 하

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.

C. 선 충 에서 신경 세포를 세균 세포를 재 설 정할 RNAi 및 열 충격 유도 하는 전사 인자 식의 적용

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)

라이브 창 꼬마 선 충 에 Lysosomal Alkalinization 평가

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.

꼬마 선 충 생식 핵의 분포, 단백질, 및 골격을 전산 분석

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).

노드 및 스캐닝 전자 현미경 검사 법, 면역 형광 산 전체를 사용 하 여 Gastrulating 마우스 배아에서 Notochordal 접시를 시각화

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.

성인 Zebrafish 부상 모델 뼈 조직 재생에 Prednisolone의 효과 연구

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.

자외선-C (UV-C) 손상에 응하여 1 차 마우스 안구 표면 세포/줄기 세포에 있는 산화 손상의 평가

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