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><tbody><tr><td>Acetate buffer</td><td>*</td><td>*</td><td>To make 100 ml: 4.8 ml 0.2 M Acetic acid, 45.2 ml 0.2 M Sodium acetate &amp;amp; 50 ml 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 pH 2.5</td><td>*</td><td>*</td><td>To make 500 ml: 15 ml acetic acid, 5 g Alcian Blue &amp;amp; 485 ml 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 &amp;ndash; store at -20 &amp;deg;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 500 ml: 50 ml glycerol, 50 ml Tris 0.1M pH8.8, 100 ml EtOH, 300 ml saline (0.9% NaCl in water), DTT 1.7 g.&amp;nbsp; Demucifying solution can be made in advance and stored, but DTT sholud be added just before incubation (340 mg/100 ml).</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&amp;nbsp;</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>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</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>&amp;nbsp;5&amp;times; 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 min. 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.&amp;nbsp;</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 MgCl&lt;sub&gt;2&lt;/sub&gt;, 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>0.5 M NaCl, 10 mM 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 &amp;micro;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 100 ml: 1 g Hydroquinone, 5 g sodium sulphite &amp;amp; 100 ml 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 100 ml: 10 ml Acetate buffer, 87 ml distilled water, 3 ml 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 &amp;ndash; store at -20 &amp;deg;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 1x PBS</td></tr><tr><td>X-gal stain</td><td>*</td><td>*</td><td>X-gal stain; 200 &amp;#956;l X-gal (A) in 50 ml solution B (0.214 g MgCl2, 0.48 g K-ferricyanide, 0.734 g K-ferrocyanide in 500 ml PBS).&amp;nbsp; Solution B can be made up oin advance and stored at 4 &amp;deg;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 ...

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Universidad de Cartagena UDEC

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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

17.6K 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

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

Morphogenesis 배아 및 Perinatal 대동맥의 병 인을 공부 하 고 Vivo에서 , 조직 및 세포 Explant 접근을 사용 하 여

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

연구

발생학

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

냉동 절편을 위한 자기 나노입자 전착 바이러스성 벡터를 사용하여 1 차적인 마우스 장 오르가노이드의 유전 공학

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

생체 외 장내 줄기세포 기능을 연구하기 위한 쥐 결장 음와의 3차원 배양

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

암 연구학

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

장 줄기와 틈새 세포의 기능 분석을 위한 Organoid 재구성 분석 결과 (오 라)

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

생체공학

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

표준화된 장 루프 모델을 사용하여 마우스에서 장 투과성 및 호중구 환전 이동의 기능 평가

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

면역학 및 감염병학

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

뮤린 소장 상피 오가노이드와 선천적 림프성 세포의 공동 배양

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

면역 조직 화학적 및 면역 형광 분석을위한 창자 조직 준비를위한 향상된 스위스 압연 기술

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

생물학

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

이온화 조사에 대한 반응으로 장 상피 재생

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

확립된 쥐 대장에 대한 장 염증의 상피 효과

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

저자 스포트라이트: 확립된 쥐 결장체에 대한 장 염증의 체외 상피 효과 연구  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

확립된 쥐 콜로노이드에 대한 시험관 내 장 염증의 상피 효과 연구

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

조건을 사용하여 쥐 소장에서 재생성 Paneth 세포의 역할을 분석하기위한 프로토콜

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Morphogenesis 배아 및 Perinatal 대동맥의 병 인을 공부 하 고 Vivo에서 , 조직 및 세포 Explant 접근을 사용 하 여

냉동 절편을 위한 자기 나노입자 전착 바이러스성 벡터를 사용하여 1 차적인 마우스 장 오르가노이드의 유전 공학

생체 외 장내 줄기세포 기능을 연구하기 위한 쥐 결장 음와의 3차원 배양

장 줄기와 틈새 세포의 기능 분석을 위한 Organoid 재구성 분석 결과 (오 라)

표준화된 장 루프 모델을 사용하여 마우스에서 장 투과성 및 호중구 환전 이동의 기능 평가

뮤린 소장 상피 오가노이드와 선천적 림프성 세포의 공동 배양

면역 조직 화학적 및 면역 형광 분석을위한 창자 조직 준비를위한 향상된 스위스 압연 기술

이온화 조사에 대한 반응으로 장 상피 재생

확립된 쥐 콜로노이드에 대한 시험관 내 장 염증의 상피 효과 연구

확립된 쥐 콜로노이드에 대한 시험관 내 장 염증의 상피 효과 연구