실험실에서 작업은 NSF (MCB0919933)와 NIH (GM088790)에 의해 지원됩니다.

1. 트렁크의 조각 재생 분석 <ol><li> 동물 폐기물 및 섭취 식품 (~ 30 그대로 웜, 각 벌레 ~ 포스트 기아 길이 80~10mm) 무료입니다 보장하기 위해 재생 분석하기 전에 최소한 5 일 동안 벌레의 일대 먹이를 중지합니다. </li><li> 분석의 날, 물을 미리 냉동 파괴 얼음 접시를 씻어 및 플라스틱 포장으로 평면 아이스 표면을 커버. 포셉를 사용하여 플라스틱 랩에 하나의 필터 용지를 넣으십시오. </li><li> 물이 몇 방울로 필터 종이를 축축하게하다. 전송 피펫을 사용하여 필터 (필터마다 &lt;20 웜)에 planarians을 전송합니다. 웜 더 샘물과 repipetting하여 필요한 경우, 필터에 재배치 수 있습니다. 초과 액체를 제거합니다. </li><li> 메스를 사용하여 동물의 앞쪽에 정점과 인두 (그림 1A)의 앞쪽에 끝 사이의 약 절반을 만든 싱글 컷으로 머리를 절단. </li><li> 메스를 사용하여 약 절반 꼬리 동물의 종료와 인두 (그림 1A)의 후부 끝 사이에 만들어진 하나의 상처로 꼬리 지역을 절단. 절단 후 70 % 에탄올로 moistened 종이 타월을 사용하여 메스에 과도한 가래를 제거합니다. 메스에서 초과 에탄올을 닦아주십시오. </li><li> 샘물이 들어있는 페트리 접시 (100 X 25mm 깊이)에 집게를 통해 필터를 전송합니다. (상처의 봉합, 상처의 곤란과 고난으로 관찰할 수) ~ 3 분 동안 기다리십시오. </li><li> thermostatted 인큐베이터로 재생 분석 및 전송 (24 ° C)에 원하는 매체를 포함하는 페트리 접시에 여과지에서 간선 조각을 씻어. </li><li> 재생 구조는 (그림 1A) 확인할 수있다 ~ 1 주일 만에 점수가 재생 표현형. </li></ol> 2. praziquantel에 의해 유도된 bipolarity : 중생의 약리 조작 <ol><li> DMSO의 solubilizing 약물 (200mM 주식 1mL에 62.4mg PZQ)에 의해 praziquantel (PZQ) 재고를 준비합니다. 당일에 사용하십시오. </li><li> 버퍼 Montjuch 소금에서 마약이 포함된 솔루션 (90 μm의 PZQ)의 50mL를 확인합니다. 분산을 위해 ~ 1 분 와동. </li><li> [프로토콜 # 1] 최종 단계 [1.7]에서 PZQ에 트렁크 조각의 코호트를 노출, 위의 내용과 같은 트렁크 조각 재생 분석을 수행합니다. </li><li> 24-48시간 후, 교환 PZQ 함유, 샘물에 대한 미디어 웜 여러 (&gt; 3) 번 세척. </li><li> 인큐베이터에 벌레를 반환합니다. 점수 bipolarity (2 헤드, 그림 1B) 절단 후 일주. </li></ol> 3. 중생의 유전자 조작 : 생체내 RNAi 먹이 프로토콜 <ol><li> 이전 분석하기 위해 닭고기 간 homogenate를 준비합니다. foodstock에서 지방산, 노란색 컬러 섹션, 그리고 퓌레 나머지 간을 폐기하십시오. 저장 메쉬 스트레이너 및 나누어지는 서스펜션 (1.5mL 튜브, -20 ° C)를 통해 필터 퓌레. </li><li> 이전 분석하기 위해, -20 ° C.에 저장된, 1mL 샘플로 공급을 aliquoting하여 소 적혈구 (RBCs)를 준비 </li><li> RNAi에 대한 웜을 선택합니다. 세 동료가 사용됩니다 : 관심의 유전자, 긍정적인 제어 (알려진 RNAi의 표현형과 유전자, 그림 1C, D &amp; E)과 부정 제어 (NO RNAi의 표현형과 유전자, 실험 코호트에 비해 최저 수준의 평가에도 유용합니다.) 각 코호트 들어, ~ 250 벌레를 (최소한 5 일 굶어 후 긴 ~ 80~10mm)을 사용합니다. 플라스틱 욕조에 샘물에 보관하십시오. </li><li> 관심 dsRNA 대상으로 유전자를 표현 E. 대장균의 각 RNAi의 보병대, 해동 재고에 대한 [8], 닭고기 간 homogenate의 튜브와 함께 [3.1]와 RBCs의 튜브 [3.2]. </li><li> 펠렛 박테리아 (1만3천그램 1 분). 2xYT 미디어 (~ 700μl)에 뜨는 및 resuspend 펠렛을 제거합니다. 얼음 뜨는, 장소 펠렛을 삭제, Recentrifuge. </li><li> 팁의 끝을 잘라하여 큰 구멍 P200 피펫 팁을 만듭니다. 철저히 RNAi 수유 믹스를 만들 수 닭고기 간 homogenate (150 μL)와 RBCs (50 μL)의 혼합물에있는 박테리아 펠렛을 resuspend. 원심 분리하여 기포를 제거합니다. </li><li> 먹이를 들어, (~ 1 인치 깊이를 떠나) 벌레가 들어있는 플라스틱 욕조에서 물의 대부분을 제거합니다. 소용돌이 욕조는이 컨테이너의 중심에 벌레를 집중하고, 벌레를 corralling 원안에 피펫 RNAi의 먹이 혼합합니다. 신중하게 다른 식사를 perturbing 않고 RNAi의 먹이 혼합에 다시 escapees을 동축 케이블로 전송 피펫을 사용! </li><li> 수유 후 (~ 1 시간), 잘 공급되는 planarians를 확인합니다. 이 벌레는 섭취 RBCs에서 딥 레드 착색을 보여줍니다. 다른 사람을 폐기하십시오. </li><li> 신중하게, 신선한 샘물과 솔루션을 먹이 음식 egestion을 방지하기 위해 교란을 최소화 바꿉니다. 이렇게하려면 부드럽게 전송 피펫을 사용하여 튜브의 한쪽으로, 흐린 물을 부어하고, 조심스럽게 욕조의 반대편 벽을을 붓고 신선한 물로 교체 벌레를 집중. Resubmerge의 planarians 신속하게 공기에 장기간 노출로도 식품 egestion 결과. </li><li> 대충 SE앨 컨테이너 뚜껑과 인큐베이터로 돌아갑니다 (24 ° C) </li><li> 재생주기 [프로토콜 # 1] RNAi의 phenotypes에 대한 화면으로 함께 interspersed 여러 사이클을 통해 RNAi 먹이 프로토콜 (따로 ~ 2-3일)를 반복합니다. 많은 유전자에 대한 효과적인 이송 및 재생을위한 표준 프로토콜은 총 기간이 ~ 1 달 걸립니다 그림 2에 표시됩니다. </li></ol> 최적의 프로토콜과 같은 mRNA의 안정성, 조직 현지화, 단백질 perdurance, 또는 재생 후 여러 먹이주기 걸로 표현형의 개발과 같은 요인에 따라 달라집니다로, 다른 유전자에 대해이 제도를 수정합니다. 단순히 부정적인 제어 일대와 타겟 mRNA 수준을 비교하는 (명백한 경우) 표현형의 penetrance에 대한 심사 또는 qPCR의 방식에 의해 최저 수준을 평가합니다. 4. 대표 결과 : 트렁크 조각 재생 분석은 모든 벌레 정상 앞쪽에 - 후부 ( &#39;꼬리에 머리&#39;) 극성을 재생성해야하는 등 강력합니다. 이것은 바로 앞쪽에 eyespots (asterixed, 그림 1A)의 출현에 의해 용이 절단 후 5 일,로 득점 수 있습니다. 그러나, 손실 구조의 완전한 형태학의 복원은 일주일 뒤에 발생합니다. 두 달린 벌레를 항복하는 PZQ하여 트렁크 조각 재생의 약리 조작 (그림 1B)가 또한 강력합니다 (EC 50 = 87 (+ -) 11 %의 바이폴라 조각 48 시간 20 70μM PZQ). Bipolarity 하나의 재생 사이클을 통해 발생합니다. 이러한 assays의 낮은 효율 가능성이 약물 용해도 및 / 또는 저장하거나, PZQ 안 먹힐 다른 flatworm 종의 사용 문제와 관련이 있습니다. 낮은 penetrance과 마약, anteriorization 지수는 종종 전체 bipolarity의 22 별도로 중간 phenotypes을 점수하는 데 사용됩니다. 긍정적인 제어 구조의 RNAi로 인한 Phenotypes는 그림 1에 표시됩니다 : 자포니카 Dugesia의 RNAi 여섯 1 (D J - 6 - 1) 눈이없는 표현형 23 일 (그림 1C), Dugesia 자포니카 PC2의 RNAi (DJ - PC2) 결과 빛이 싫다고 응답 24 (그림 1D)과 RNAi의 손실 결과 Dugesia 자포니카 βcatenin - 1 (DJ - βcatenin - 1) 트렁크 조각 (그림 1E)에서 두 개의 머리 표현형 16-19, 21을 초래합니다. 이러한 phenotypes은 다음 간단한 RNAi 프로토콜을 사용하여 얻을 수있다 : DJ - 6 - 1 (FFxFx), DJ - PC2 (FFFx), DJ - βcatenin - 1 (FFxFx) 어디 F = 먹이주기 및 X = 재생 사이클 각각. <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="그림 1"/> 그림 1 :... 트렁크 조각 재생 분석 왼쪽, excised 트렁크 조각의 위치를 표시 planarian (위)와 개략도 (중간)의 이미지를 마우스 오른쪽, 트렁크 조각 재생의 timecourse이 표시된 시간에 재생 조각의 모양 (일) 보여주 B PZQ 노출에 의해 제작. 쌍두 planarian 이미지 DJ - 6 - 1 RNAi. 모바일 컨트롤 벌레에 비해 고정되어 DJ - PC2 RNAi 웜 (스테인드 적색)의 빛을 혐오 반응의 D 손실 (스테인드에 의해 만들어진 C &#39;눈이없는&#39;벌레 녹색). E 바이폴라 planarian는 DJ - βcatenin - 1 RNAi에 의해 만들어진. BE에서 RNAi 벌레의 원래 앞쪽에 끝이 왼쪽으로 지향합니다. <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="그림 2"/> 그림 2 : D. 많은 유전자의 분해에 대한 효과가 여러 feedings로 구성된 전형적인 RNAi 프로토콜 (F) 및 재생 사이클 (X)에 대한주기를 먹이 및 절단의 순서 자포니카. 개별 유전자에 대해이 프로토콜의 변경은 사용하는 예를 들어, 최적의 결과를 얻을 필요가있을 수 있습니다 적은 (괄호) 또는 수유 및 재생 사이클의 큰 수입니다.

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S., S, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+biological+activity+of+praziquantel+requiring+voltage-operated+Ca2++channel+beta+subunits:+subversion+of+flatworm+regenerative+polarity.">A novel biological activity of praziquantel requiring voltage-operated Ca2+ channel beta subunits: subversion of flatworm regenerative polarity.</a> PLoS Negl Trop Dis. 3, e464-e464 (2009).</li><li>Oviedo, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+neural+and+gap+junction+protein-mediated+cues+control+polarity+during+planarian+regeneration.">Long-range neural and gap junction protein-mediated cues control polarity during planarian regeneration.</a> Dev Biol. 339, 188-199 (2010).</li><li>Nogi, T., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+innexin+gene+expression+and+functional+roles+of+gap-junctional+communication+in+planarian+regeneration.">Characterization of innexin gene expression and functional roles of gap-junctional communication in planarian regeneration.</a> Dev Biol. 287, 314-335 (2005).</li><li>Mannini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Djeyes+absent+(Djeya)+controls+prototypic+planarian+eye+regeneration+by+cooperating+with+the+transcription+factor+Djsix-1.">Djeyes absent (Djeya) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1.</a> Dev Biol. 269, 346-359 (2004).</li><li>Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+planarian+central+nervous+system+(CNS)+revealed+by+neuronal+cell+markers.">Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers.</a> Zoolog Sci. 15, 433-440 (1998).</li></ol>

관심 없음 충돌 선언하지 않습니다.

프로토콜은 자포니카 planarian의 Dugesia의 재생을 연구하고 조작 여기 상세 assays을 설명했다. 그들은 간단하고 쉽게 실험실이나 교실에서 수행할 수있는 전문 장비가 이러한 요구하지 않습니다. Assays 개별적으로 수행하거나, 결합 (생체내에서 마약 efficacies의 화학 유전자 검사)와 후보 유전자 수준에서 수행할 수 있습니다, 또는 불편, 높은 처리량 검사 8 적응력이 아르 수 있습니다. 자신의 권리를 planarians의 흥미로운 생물학을 공부에 대한 여부, 또는 조직 재생을 연구에 대한 대안 모델의 포유 동물 homologues의 생체내 기능의 평가에 대한 이러한 접근 방식은 연구자의 ​​다양한에서 관심을 catalyze해야합니다.

<table border="1"><tbody><tr><th> 시약 </th><th> 공급 업체 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 스프링 워터 </td><td> Kandiyohi. 프리미엄 워터스 주 미니 애폴리스, MN </td><td> N / A </td><td> 샘물의 다른 형태도 잘 작동합니다. 생존의 assays의 첫 번째 시련. </td></tr><tr><td> NaCl (1.6mM), CaCl2 (1mM), MgSO4 (1mM), MgCl2 (0.1mM), KCl (0.1mM), NaHCO3 (1.2mM), HEPES (1.5mM) : 1 x는 Montjuïch 소금을 버퍼. 24 산도 7.4 ° C. </td><td> 여러 공급 업체 </td><td> N / A </td><td> 산도 버퍼링을 보장하기 위해 재생 assays 동안 약물 치료를위한 인공 물. 8분의 5 Holtfreter의 솔루션은 대안입니다. </td></tr><tr><td> 2xYT 맛을 </td><td> 피셔 사이 언티픽 </td><td> BP2467 - 500 </td><td> 미디어 = 31g / L. Autoclaved. </td></tr><tr><td> 페트리 접시 (100x25mm) </td><td> 피셔 사이 언티픽 </td><td> 08-757-11 </td><td> 재생주기 동안 주택 웜 </td></tr><tr><td> 스퀘어 접시 (100x100x15mm) </td><td> 피셔 사이 언티픽 </td><td> 08-757 - 11A </td><td> 벌레 절단 표면으로 사용 얼음 트레이를위한 물, 동결로 채우기 </td></tr><tr><td> 플라스틱 욕조 : Ziploc 트위스트 &#39;N은 Loc (16온스). </td><td> 다양한 소매 </td><td> N / A </td><td> RNAi의 동료를위한 편리한 물을 꽉 용기 </td></tr><tr><td> 치킨 간 </td><td> 상업 식료품 </td><td> N / A </td><td> 항생제의 소지를 없애기 위해 때문에 유기 공급을 향해 편견. </td></tr><tr><td> 핸드 블렌더 </td><td> 모든 부엌 공급 </td><td> N / A </td><td> 닭고기 간 퓨레를 만들기위한 사용 </td></tr><tr><td> 와이어 1mm 메쉬 스트레이너 </td><td> 모든 부엌 공급 </td><td> N / A </td><td> 닭고기 간 퓨레를 긴장에 사용 </td></tr><tr><td> 보빈 적혈구 </td><td> Lampire 생물학 연구소 </td><td> 7,240,807 </td><td> 백퍼센트 RBC 서스펜션을 씻었 풀링 </td></tr><tr><td> 서큘러 여과지 </td><td> 왓먼 # 3 </td><td> 1003 055 </td><td></td></tr><tr><td> 전송 피펫 </td><td> 피셔 사이 언티픽 </td><td> 13-711-41 </td><td></td></tr><tr><td> 멸균, 수술 블레이드 </td><td> 여러 공급 업체 </td><td> N / A </td><td></td></tr><tr><td> ± Praziquantel </td><td> 시그마 알드리치 </td><td> P4668 </td><td> 4 어둠 속에 저장 파우더 aliquots ° C. 마르다. </td></tr><tr><td> DJ - 6 - 1 </td><td></td><td> GenBank AJ557022.1 </td><td> &quot;눈을 이하&quot;표현형 23 RNAi 긍정적인 제어 </td></tr><tr><td> DJ - βcatenin - 1 </td><td></td><td> GenBank AB181913.1 </td><td> 두 머리 표현형 17 RNAi 긍정적인 제어 </td></tr><tr><td> DJ - PC2 </td><td></td><td></td><td> 가벼운 혐오의 표현형 24 손실 RNAi 긍정적인 제어 </td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

자유 생활 planarian의 flatworms 자신의 뛰어난 재생 능력을 1로 실험적인 사용 때문에 오랜 역사를 가지고. 이 동물에서 excised 작은 조각이 없거나 신체 구조의 재생 다음 원래 신체 계획을 개혁. &#39;트렁크&#39;조각이 손상 웜으로부터자를 경우 예를 들어, 새 &#39;머리&#39;anteriorly 재생되며, &#39;꼬리&#39;는 (그림 절단하기 전에 원래의 신체 구조의 &#39;머리 - 투 - 꼬리&#39;극성을 복원 뒤로 재생성합니다 1A). 재생 정상 신체 항상성 및 집행 조직 재생 중에 ~ 30 서로 다른 세포 유형으로 차별 &#39;neoblasts&#39;로 알려진 planarian 줄기 세포에 의해 구동됩니다. 이 재생 과정은 강력하고 입증하기 쉽습니다. 여러 선구 실험실, 다양한 도구와 기능 유전 방법의 헌신에 때문에 지금은이 모델을 시스템에 최적화된되었습니다. 따라서, 상당한 최근의 진전은 planarian 발달 소성 2-9을 underpinning 분자 이벤트를 이해하고 조작하였습니다. planarian 모델 시스템은 과학자들의 광범위한 관심을 것입니다. neuroscientists 들어, 모델은 전체 신경계의 재생을 연구보다는 단순히 regrowth / 일반적으로 많이 설립 모델에 연구의 초점 아르 단일 신경 세포 프로세스의 복구합니다. 수있는 기회를 내실수 Planarians는 신경 전달 물질 10 과다을 표현 중추 신경계 11, 12의 진화를 연구를위한 중요한 시스템을 대표하고 행동 선별 가능성 13, 14 있습니다. 재생 결과는 약리 및 유전자 조작에 의해 apparoaches 의무가 있습니다. 예를 들어, 약물 단순히 몸과 조각을 배치 약물이 포함된 절단 후 다른 시간 지점에서 솔루션을하여 재생에 대한 효과에 대한 심사 수 있습니다. 개별 유전자의 역할은 microinjection의 사이클이나 dsRNA 구조 8, 9, 15 bacterially - 표현 이송을 통해 얻을 수있는 최저의 방법을 (생체내 RNAi)를 이용하여 공부하실 수 있습니다. 두 방식은 바이폴라 동물 16-21 높은 penetrance - 예를 들어, 재생시 시각적으로 눈에 띄는 phenotypes를 만들 수 있습니다. 이 모델과 같은 방법을 구현 채택을 촉진하기 위하여, 우리는 planarian의 Dugesia가 자포니카 사용 약리 및 유전 assays (생체내 RNAi의 먹이에 의해)이 동영상 기사 프로토콜의 쇼케이스.

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

살아있는 동물 이내에 줄기 세포 차별을 공부에 대한 매력적인 모델은 planarian의 flatworm입니다. 재생 쉽게 기본적인 실험실에서 수행하는 간단한 절단 실험에서 공부하고 약리 및 유전 (의무뿐입니다<em&gt; 생체내에</em&gt; RNAi)이 문서의 프로토콜에 의해 내용으로 조작.

약리 및 기능성 유전자 Assays은 Planarian의 재생을 조작하는<em자포니카> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

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Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.7K 조회수.  University of Minnesota Medical School. 살아있는 동물 이내에 줄기 세포 차별을 공부에 대한 매력적인 모델은 planarian의 flatworm입니다. 재생 쉽게 기본적인 실험실에서 수행하는 간단한 절단 실험에서 공부하고 약리 및 유전 (의무뿐입니다 생체내에 RNAi)이 문서의 프로토콜에 의해 내용으로 조작.

비디오: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

마우스에 대한 진한 / 라이트 전환 테스트

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

연구

생물학

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

피질 Neurogenesis : 세포 기반 치료하기 위해 연구소의 발전에서 전환

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

칙의 배아에서 신경 유도에 대한 분석

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

마우스에 대한 고가 플러스 메이즈

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

성인 마우스 Hippocampal 이가있는 이랑의 해부

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Embryogenesis 동안 중복 기능을 테스트하기 위해 역방향 유전자 접근

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

유전자 협회의 기능 기초를 테스트하는 Allele 특정 유전자 발현 분석

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planarian의 부동화, 부분 조사 및 조직 이식

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

에 박테리아 기능 네트워크 및 경로를 매핑<em&gt; 대장균</em&gt; 합성 유전자 배열을 사용하여

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

유전자 표현 기계의 기능 Assays을위한 소규모 핵 추출물

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

약리 및 기능성 유전자 Assays은 Planarian의 재생을 조작하는 Dugesia