This study was supported by NIH grant DE025014 to JQF.

모든 프로토콜을 검토하고 텍사스 A &amp; 치과 M 대학에서 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 1. 동물 사육 <ol><li> 이 연구에서 세 동물 모델을 사용합니다. 먼저 사용 Col10a1-Cre 호텔 15 쥐 외과 형성의 배아 연골 세포의 운명을 조사하고 Rosa26 tdTomato로 교차합니다 (B6, 129S6- GT는 (ROSA) 26Sor TM9 (CAG-tdTomato) Hze / J) 마우스를 Col10a1-를 얻기 위해 CRE와 Rosa26 tdTomato 쥐. 다음으로, 2.3Col1a1-GFP 마우스 16이 쥐를 교차. Col10a1-Cre 호텔을 사용합니다; Rosa26 tdTomato; 2.3Col1a1-GFP 마우스는 셀 혈통 추적 실험 (그림 1B)를 수행 할 수 있습니다. </li><li> 출생 후 연골 세포의 운명을 연구하기 위해, 단계 1.1에서와 동일한 절차를 수행하지만, Aggrecan-CRE의 ERT2 (AGG-Cre 호텔 ERT2를 사용) 17, 18 라인과 AGG-Cre 호텔 ERT2을 유지; Rosa26 tdTomato; 2.3Col1a1-GFP 마우스는 세포 계보 추적 실험 (도 1C)을 수행한다. 생후 14 일에 타목시펜을 주입한다. </li><li> 면역과 기술을 추적 세포 혈통을 결합하려면, AGG-Cre 호텔 ERT2 마우스와 Rosa26 tdTomato 마우스를 교차. 실험에 모두 유전자를 가지고 마우스를 사용하여 출생 후 3 일 (그림 1D)에 타목시펜을 주입. </li></ol> 2. 재료 준비 <ol><li> 10 ㎎ / ㎖의 농도에서 10 % 에탄올 및 90 % 옥수수 유에서 타목시펜 분말을 녹인다. 주사 투여 량은 75 ㎎ / ㎏이다. </li><li> 7.4의 pH 값을 조정하기 위해 2 M 나트륨 수화물을 사용하여 4 %의 농도로 PBS에 파라 포름 알데히드 (PFA) 분말을 녹인다. 희생 후 조직 (하악 외과와 뒷다리가 여기에 사용되는)를 해결하기 위해 4 % PFA 솔루션을 사용합니다. 때문에 자사의독성, 장갑과 안면 마스크와 후드에 PFA를 처리합니다. </li><li> 7.4의 pH 값을 조정하기 위해 2 M 나트륨 수화물을 사용하여 10 %의 농도로 증류수에 EDTA 분말을 녹인다. 하악 외과와 뒷다리를 석회 10 %의 EDTA를 사용합니다. </li><li> 15 %와 30 %의 농도로 PBS에서 자당 분말을 녹여. 탈회 후 조직을 탈수 자당 솔루션을 사용합니다. </li><li> 2 ㎎ / ㎖, pH 5.0으로의 농도로 PBS에 히알루 분말을 녹인다. 이 솔루션은 면역 항원 검색을위한 것입니다. </li><li> Runx2 또는 DMP1 면역 염색을 위해 PBS로 3 % 소 혈청 알부민 (BSA), 20 %의 염소 혈청이 포함 된 차단 용액을 제조 1.5mL 용량 튜브를 사용한다. </li><li> Runx2 또는 DMP1 면역 염색을 위해 PBS로 2 % 염소 혈청을 포함하는 일차 항체 용액을 제조 1.5mL 용량 튜브를 사용한다. Runx2 400 및 1 : 100 DMP1 대한 일차 항체의 농도는 1이다. </li><li> 제조 1.5 ㎖의 튜브를 사용하여거짓 긍정적 인 결과를 피하기 위해 면역 염색에 대한 제어와 토끼 IgG의 솔루션입니다. 이 솔루션은 PBS의 2 % 염소 혈청이 포함되어 있습니다. Runx2 제어 토끼 IgG의 농도는 1 : 400; 100 : DMP1, 1. 동시에 실험 및 제어 염색을 수행합니다. </li><li> Runx2 또는 DMP1 면역 염색을 위해 PBS로 2 % 염소 혈청을 포함하는 이차 항체 용액을 제조 1.5 ㎖의 튜브를 사용한다. 500 : 1의 희석에 차 항체를 사용합니다. </li></ol> 3. 슬라이드 공 초점 현미경을위한 준비 (만 추적 휴대 리니지, 면역 형광 아니오 조합) <ol><li> 유리한 시점에서 AGG-CRE ERT2 마우스에서 타목시펜을 주입한다. <ol><li> 첫째, 케이지에서 마우스를 제거합니다. 그런 다음, 복부를 노출, 마우스의 뒷면에있는 피부를 잡아 뒤집 왼쪽 엄지와 검지 손가락을 사용합니다. 주사기를 잡고 오른손을 사용합니다. 주입을위한 최적의 엔트리 포인트는 L에EFT 또는 hypogastrium의 오른쪽, 간 및 방광을 피하는. </li><li> 마우스의 뒷다리에 주사기 평행을 유지하고 복강 주사. 주사 투여 량은 75 ㎎ / ㎏이다. 각각 18g - 9g, 11 - - 13g, 16 이주 3 주, 그리고 옛 사주의 나이에 마우스의 무게는 약 7이다. </li></ol></li><li> 크 실라 / Ketaset 조합으로 쥐를 마취. <ol><li> 작동 용액을 제조하기 위해, 각각 제 1 ㎎ / ㎖, 5 ㎎ / ㎖의 농도로 증류수 및 크 실라 Ketaset 희석. 이 비율 다음으로, (1)의 크 실라와 Ketaset 섞는다. 주사 투여 량은 30 μL / g이다. </li><li> 단계 3.1에서와 같이 주입한다. 마우스의 발목을 곤란하게하여 마취를 확인합니다. 그것은 아무 반응이없는 경우 마우스가 의식이다. </li></ol></li><li> 마취 후 4 % PFA와 마우스를 관류. <ol><li> 마우스가 의식을 잃게 한 후, entirel하기 위해 보드에 마우스의 4 개의 다리를 고정y는 복부를 노출합니다. </li><li> 70 % 에탄올로 복부를 포화 중간 선을 따라 목에 하복부에서 절제를합니다. 복막 멤브레인을 나타 내기 위해 측면에 피부를 당겨 동시에 핀치합니다. 길이 절단을 만들기 위해 해부 가위를 사용합니다. </li><li> 잘라와 마음을 노출 전면 갈비뼈를 제거합니다. 오른쪽 귓바퀴에 슬롯을 잘라 동시에, 22 G 주사기와 좌심실에서 심장에 구멍 주사기를 개최합니다. </li><li> 천천히 심혈을 따라 관류하는 4 % PFA를 주입하는 동안 오른쪽 귓바퀴에서 컷 아웃 혈액 플러시. 관류를위한 PFA의 부피 1 ㎖ / g이다. 하드 덕트 건물의 배기 시스템에있는 클래스 I의 바이오 안전성 캐비닛이 단계를 수행합니다. </li></ol></li><li> 마우스의 피부를 벗겨 및 4 ℃에서 하룻밤 해결하기 위해 4 % PFA 40 ml에 포함 된 50 ml의 폴리 프로필렌 원심 분리 튜브에 몸 전체를 넣어. </li><li>조심스럽게 몸에서 하악과 뒷다리를 제거하는 해부 가위와 # 3, # 5 집게를 사용하여 표면에 근육과 힘줄을 제거 할 수 있습니다. 하드 덕트 건물의 배기 시스템에있는 클래스 I의 바이오 안전성 캐비닛이 단계를 수행합니다. </li><li> 세 번째 몰의 말단 부위에 두 개의 조각으로 하악을 잘라. 마찬가지로, 탈회를 가속화하기 위해 골수 공동을 노출 간부의 대퇴골과 경골을 자른다. 외과와 과두 과정을 포함하는 부분을 넣고 10 % EDTA의 40 ㎖의에 뒷다리와 함께 2 4 ° C에서 석회하기 - 50 ml의 폴리 프로필렌 원심 분리 관에 사일. </li><li> 50 ml의 폴리 프로필렌 원심 분리 관에 4 ° C에서 외과 하룻밤 뒷다리를 탈수 15 % 자당의 50 ㎖를 사용합니다. </li><li> 50 ml의 폴리 프로필렌 원심 분리 관에서 밤새 4 ° C에서 외과와 뒷다리를 탈수 30 % 자당을 사용합니다. </li><li> 시상면 함께 샘플을 포함동결 절단 (Cryosections) 기계의 절삭 접시에 10월 <ol><li> 수평 외과 또는 설치 금형의 뒷다리를하다. OCT에서 조직을 잠수함과 OCT를 정지 할 때까지 동결 절단 (Cryosections) 기계에 둡니다. </li><li> 절단 접시에 OCT의 블록을 탑재합니다. OCT를 완전히 얼어 붙은 있는지 확인하기 위해 절단하기 전에 약 15 분 동안 기다립니다. </li></ol></li><li> 10 μm의 섹션으로 외과와 뒷다리를 잘라. -20 ° C에서 슬라이드 및 저장에 섹션을 수집합니다. </li><li> 염색하기 전에 물을 제거하기 위해 37 °의 C 챔버 슬라이드를 부화. </li><li> 5 분 동안 증류수로 두 번 슬라이드를 씻으십시오. </li><li> 각 섹션 주위에 물을 닦아냅니다. 섹션을 원과 원에 DAPI 또는 비 형광 안티 - 페이드 장착 솔루션을 드롭 소수성 장벽 펜을 사용합니다. 조심스럽게 커버 슬립을 누워. </li></ol> Runx2와 DMP1 4. 면역 조직 화학 염색 참고 : IgG의 콘면역 조직 화학 염색이 거짓 양성 신호를 방지하기 위해 트롤이 필요합니다. 실험 및 대조군에 대한 염색은 동시에 수행 될 필요가있다. <ol><li> 염색하기 전에 물을 제거하기 위해 37 °의 C 챔버 슬라이드를 부화. </li><li> 증류수로 두 번 슬라이드를 씻으십시오. </li><li> 슬라이드의 모든 부분에 동그라미를하는 소수성 장벽 펜을 사용합니다. 이 단계에서 완전히 섹션을 다루 원에 준비된 솔루션을 모두 추가 할 수 있습니다. </li><li> 30 분 동안 37 ° C에서 습기 챔버에서 히알루와 섹션을 처리합니다. (단계 4.5 - 4.8) 용액의 부피는 부분의 크기에 의존한다. 긴 뼈의 외과 용 용액 50 ㎕를 100 μl를 사용합니다. (0.1 % 함유 PBS 트윈 20) PBST로 3 회 씻는다. </li><li> 준비하고 각 섹션에 블로킹 용액을 적용하고, 실온에서 1 시간 동안 습윤 챔버에이를 부화. </li><li> 차와 섹션을 품어밤새 4 ° C에서 항체 용액 (토끼 항 - 마우스의 Runx2, 또는 토끼 항 - 마우스 DMP1). PBS로 3 회 세척한다. </li><li> 실온에서 2 시간 동안 차 항체 용액 (염소 항 - 토끼 알렉사 형석 488)와 섹션을 인큐베이션. PBS로 3 회 세척한다. </li><li> 절 주위에 물을 닦아 슬라이드 섹션을 다루 원에 DAPI 놓습니다. 조심스럽게 커버 슬립을 누워. </li></ol> 5. 공 초점 현미경 <ol><li> 488 μm의 (녹색)에서 561 μm의 (적색)에 이르기까지 다양한 파장에서 공 초점 현미경을 사용하여 형광 세포 이미지를 캡처합니다. 10X, 20X 및 63X 렌즈를 사용하여 19 200 Hz에서 여러 누적 된 이미지를 가지고 (1,024 × 1,024의 치수). </li></ol>

<ol>
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The authors declare that they have no competing financial interests or other conflicts of interest.

기술적 한계로 인해, 생체 내에서 세포의 거동을 조사하는 것이 어렵다. 그러나, 셀 혈통 추적 기술은 세포 생물학 7-9을 연구하기위한 강력한 도구로 증명된다. 본 연구에서는 상기 면역과 조합하여 프로토콜을 향상시킨다. 이러한 방식으로, 세포 운명 리니지 추적의 적용을 넓혀 연관된 여러 마커에 의해 정의 될 수있다. 또한, 면역, 토마토 신호의이 공동 현지화 동시에 단독으로 ...

개발하는 동안, 연골 내 골 형성의 골격 체적의 80 % 이상을 차지한다. 널리은 비대 연골 세포의 사멸로 시작하는 것으로 생각된다. 그 후, 기본 골수의 세포 침입과 뼈 marrow- 및 골막 유래 세포 1,2에 의해 새로운 뼈 증착 다음에 혈관 신생을 시작합니다. 비대 연골 세포 운명 HCS ()는, 그러나, 수십 년 3 논쟁의 문제였다. 초기의 HC는 연골 세포 분화 경로의 종료로 간주하고, 일반적으로 아폽토시스의 HC의 궁극적 운명 것으로 생각되었다. 지금, 일부 연구자들은 적어도 일부의 HC가 살아남을 수 및 연골 뼈 형성에 기여하는 것이 좋습니다. 그들이 그 성장을 제안했지만 판 연골 세포는 미세 구조, 면역 조직 화학 염색을 기반으로 조골 세포에 transdifferentiate 수있는 능력을 가지고 있었고, 시험 관내에서, 46이 방법 w 전혀 연구되지조골 세포 계보에 연골 세포의 기여를 입증에 결정적인 감수해야. 셀 혈통 추적 기술은 세포의 운명을 연구하는보다 엄격한 방법을 제공합니다. 간단히 말하자면, 오직 셀의 특정 유형에서 발현되는 재조합 효소, 리포터 유전자의 발현을 자극한다. 이러한 방식으로,이 세포의 종류와 그 후손 영구적 7로 표시된다. Cre 호텔-에 loxP 시스템은 일반적으로 혈통 추적에 사용됩니다. CRE (재조합 효소 효소) 둘에 loxP 부위 사이 STOP 서열을 절제하고 특정 세포주 (도 1a)에서 리포터를 활성화한다. 일부의 경우, 연구자는 에스트로겐 수용체 (Cre 호텔 ERT2) (8)의 수정 된 형태로 융합 Cre 호텔을 일으키는 등 타목시펜과 같은 약물을 사용하여 Cre 호텔을 활성화 유리한 시점을 선택할 수 있습니다. 형광 기자는 극적으로 복잡성을 줄일 수 있기 때문에 실험을 추적 혈통의 표준이되었다및 8,9 추적 세포 운명의 정확성과 효율성을 향상시킨다. 그것은 쉽게 7 (도 1A)를 시각화하게 밝은 형광 단백질과 강한 표면 형광을 갖기 때문에 tdTomato 형광 리포터 중에서 최상의 선택되고있다. 시스템을 추적 Rosa26 tdTomato 계보를 사용하여, 우리 그룹과 다른 연구자의 HC 개발 10-14 동안 뼈 세포로 자신의 표현형을 변경할 수 있습니다 것으로 나타났습니다. 2.3Col1a1-GFP (조골 세포에 특정)과 면역 (뼈 세포에 특정) :이 작업을 수행하기 위해, 우리는 Rosa26의 tdtomato (유비쿼터스 식 모든 셀에서) / Cre 호텔 (연골 세포에 특정) 마우스와 조합을 추적하는 두 세트를 개발했다. 데이터는 두 가지 방법이 생체 내에서 세포의 운명을 연구하는 가능한 방법이라는 것을 보여줍니다.

<table><tbody><tr><td>Tamoxifen&amp;nbsp;</td><td>Sigma</td><td>T5648</td><td>activate the Cre event&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td>fix the sample</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Alfa Aesar</td><td>A10713</td><td>decalcify the hard tissue</td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma</td><td>S0389</td><td>dehydrate the tissue</td></tr><tr><td>Hyaluronidase from bovine testes&amp;nbsp;</td><td>Sigma</td><td>H4272</td><td>retrieve antigen for immunochemical staining</td></tr><tr><td>Bovine serum albumin</td><td>Sigma&amp;nbsp;</td><td>A3059</td><td>blocking solution&amp;nbsp;</td></tr><tr><td>primary antibody for Runx2&amp;nbsp;</td><td>Cell Signal</td><td>D1L7F</td><td>primary antibody for immunochemical staining</td></tr><tr><td>primary antibody for DMP1</td><td>provided by Dr. Chunlin Qin</td><td></td><td>primary antibody for immunochemical staining</td></tr><tr><td>anti-rabbit IgG</td><td>Sigma</td><td>18140</td><td>control for immunochemical staining</td></tr><tr><td>secondary antibody&amp;nbsp;</td><td>Invitrogen</td><td>A11008</td><td>second antibody for immunochemical staining</td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td>4583</td><td>embed the sample for frozen section</td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337</td><td>PBST</td></tr><tr><td>non-fluorescing antifade mountant</td><td>Life technologies</td><td>P36934</td><td>mounting slides</td></tr><tr><td>DAPI</td><td>Life technologies</td><td>P36931</td><td>nuclear staining</td></tr><tr><td>Hydrophobic Barrier Pen</td><td>Vector Laboratories</td><td></td><td>circle the section on the slide for for immunochemical staining&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>AnaSed</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>Ketaset&amp;nbsp;</td><td>Zoetis</td><td></td><td>anesthetization&amp;nbsp;</td></tr><tr><td>cryosection machine</td><td>Leica</td><td>CM1860 UV</td><td></td></tr><tr><td>confocal microscope</td><td>Leica</td><td>DM6000 CFS&amp;nbsp;</td><td></td></tr></tbody></table>

co-localization of cell lineage markers and the tomato signal

셀 혈통 추적 시스템이 발달 생물학 연구에서 주로 사용되어왔다. Cre 호텔 재조합 효소의 사용은 특정 세포주 모든 자손의 리포터의 활성을 허용한다. 여기, 우리가 연골 세포를 직접 긴 뼈와 Cre 호텔, Col10a1-Cre 호텔과 Aggrecan-Cre 호텔 ERT2 (AGG-Cre 호텔 ERT2) 두 종류를 사용하여 하악 외과 개발 과정에서 조골 세포와 골 세포로 변형을 입증하는 기술을 추적 세포 혈통을 사용 Rosa26 교차 tdTomato. Col10과 aggrecan 모두 연골 세포에 대한 마커를 잘 인식하고 있습니다. 이를 바탕으로, 우리는 특정 세포 마커의 발현을 면역 형광 분석하여 정의 간 세포 운명과 함께 추적하는 새로운 방법 세포 계보를 개발했다. Runx2 (초기 단계의 조골 세포에 대한 마커) 및 상아질 매트릭스 protein1 (DMP1; 말기 조골 세포 마커) 있었다연골 유래 뼈 세포 및 분화 상태를 식별하는 데 사용된다. 이 조합은 셀의 혈통 추적의 적용을 넓혀, 또한 화합물 마우스의 생성을 간소화 아닙니다. 더 중요한 것은, 부모 세포 자손의 수, 위치 및 분화 상태는 단독으로 추적 세포 혈통보다 더 많은 정보를 제공하는 동시에 표시됩니다. 결론적으로, 셀 혈통 추적 기술과 면역의 공동 응용 프로그램은 생체 내에서 세포 생물학을 조사하기위한 강력한 도구입니다.

연골 세포는 직접 하악 외과와 긴 뼈의 뼈 세포 (조골 세포와 골 세포)로 변환. Aggrecan, 연골위한 중요한 유전자는 주로 조기 성숙 연골 세포 (18)로 표현된다. 그 결과, AGG-Cre 호텔 ERT2 연령 2 주에서 타목시펜의 주입; Rosa26 tdTomato 마우스는 모든 연골 세포와 자신의 딸 세?...

Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

Co-localization of Cell Lineage Markers and the Tomato Signal

면역과 (조골 세포에 특정) 2.3Col1a1-GFP 하나 하나 (뼈 세포에 특정) : 우리는 두 / Cre 호텔이 마우스를 (특히 연골 세포에서 발현) (보편적으로 모든 세포에서 발현) Rosa26의 tdtomato의 조합을 추적 세트를 개발했다. 데이터는 골 연골 세포로의 직접적인 변환을 보여준다.

셀 리니지 마커의 공동 현지화와 토마토 신호

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

co-localization-of-cell-lineage-markers-and-the-tomato-signal

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Developmental Biology

12.1K 조회수.  Texas A&M University College of Dentistry.  면역과 (조골 세포에 특정) 2.3Col1a1-GFP 하나 하나 (뼈 세포에 특정) : 우리는 두 / Cre 호텔이 마우스를 (특히 연골 세포에서 발현) (보편적으로 모든 세포에서 발현) Rosa26의 tdtomato의 조합을 추적 세트를 개발했다. 데이터는 골 연골 세포로의 직접적인 변환을 보여준다. 

비디오: Co-localization of Cell Lineage Markers and the Tomato Signal

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

멀티 컬러 형광 기자 마우스를 사용하여 각막 세포의 리니지 추적하는 방법

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

연구

발생학

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

쌍둥이 자리 MARCM를 사용하여 세포 계보 분석 및 유전자 기능 연구

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

다른 식물 종에서 촬영 정점 Meristems의 confocal 라이브 영상

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

기자 마우스를 사용하여 IL-22 발현 림프구의 시각화

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

면역학 및 감염병학

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmal 지역화 시퀀스 Planta에 단백질의 식별

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

식물의 성적 증명서의 정확한 현지화에 대한 원위치 하이브리드화에서

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

생물학

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

의 세포 구체적인 분석<em&gt; Arabidopsis</em&gt; 형광 활성화 셀 정렬을 사용하여 잎

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

성인 마우스 뇌에서 유도성 형광 표지 줄기 세포의 계보 추적

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

신경과학

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

담배 과도 식 시스템을 사용하여 MAPK 신호 전달 통로의 공간 개편 이미징

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

이중 마커를 이용한 모자이크 분석을 이용한 대뇌 피질 개발의 계보 추적 및 클론 분석(MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...