This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

모든 실험 절차는 버지니아 기관 동물 관리 및 사용위원회의 대학에 의해 승인되었습니다. 참고도 1은 실험 과정의 개략도를 나타낸다. 난자 1. 준비 <ol><li> 사전 프라임 (X) 난 모세포의 분리에 앞서 임신 마레 혈청 성선 자극의 150 U (PMSG) 약 1 주일와 여성을 laevis의. 29 G 바늘 1 CC 멸균 주사기 등의 림프 낭에 1 ml의 150 U / ㎖의 PMSG를 주사. </li><li> 난 모세포 주입 및 난자 동물 캡 분석을위한 솔루션을 준비합니다. <ol><li> 칼슘을 준비 ++ / 마그네슘 + + - 무료 OR2 (OR2-) : 82 mM의 염화나트륨, 2.5 밀리미터의 KCl, 1.5 밀리미터 나 2 HPO 4, 50 mM의 HEPES pH가 7.2. </li><li> OR2- 플러스 1 mM의 염화칼슘 2, 1 밀리미터의 MgCl 2 : OR2를 준비합니다. </li><li> 60 % 라이 보 비츠 L15, 0.4 ㎎ / ㎖ BSA, 100 μg의 / ㎖ 겐타 마이신, pH가 7.8 : OCM을 준비합니다. </li><li> 사전1X MBS 깎다 : 88 mM의 염화나트륨, 1 mM의 KCl을을 0.7 mM의 CaCl2를, 1mM의 황산, 5 mM의 HEPES (PH 7.8), 2.5 mM의 NaHCO3을. </li><li> 1X MBS의 3 % 피콜을 준비합니다. </li><li> 1X NAM을 제조 : 110 mM의 염화나트륨, 2 밀리미터의 KCl, 1 mM의 칼슘을 (NO 3) 2, 1mM의 황산, 0.1 mM의 EDTA, 1mM의 NaHCO3을, 2 mM의 나트륨 3 PO 4, pH가 7.4. </li></ol></li><li> 0.1X MBS 에틸 -3- 아미노 벤조 에이트, 메탄 술폰산 염을 0.03 % 용액 (MS222)으로 여성 마취위한 용액 개구리 배치하여 (제 10 ml의 95 % 에탄올 중의 0.3 g의 MS222을 용해하고 1 L 0.1X의 MBS에 희석) 10-15 분 또는 응답하지 않는 때까지. <ol><li> 그 마취가 응답하지 않도록 (불완전 마취 된 개구리가 사지를 움직이거나 몸을 뒤집)하기 위해 다시에 마취 된 개구리를 돌려 완료 확인합니다. </li></ol></li><li> 수술 집게로 난소 조직을 분리, 메스 블레이드와 피부 및 신체 벽을 통해 복부 절개를하여 난소 조각을 분리가위. OR2-에 난소 조각을 놓습니다. 24mm 곡선 바늘에 3-0 실크 봉합사와 신체 벽을 닫고 같은 봉합 별도로 피부를 닫습니다. 물에 1g / L 수족관 소금에 여성의 복구를 허용합니다. </li><li> (- 20 난자 10) 조각과 신선한 OR2-로 전송 작은으로 미세 집게, 눈물 난소 조직을 사용. </li><li> 1 시간 동안 진탕 기에서 부드럽게 교반 신선한 콜라게나 제에 전송하고 하나의 추가 시간 동안 교반하여 OR2- 2.0 ㎎ / ㎖ 콜라게나 제에 Defolliculate 난소 조각. </li><li> 워시 작은 난자를 폐기 OR2에 [칼슘 + + / 마그네슘 + +를 ​​포함하는 10 번, 난자. </li><li> OCM에 두 번 난자를 세척하고 신선한 OCM에 전송합니다. 육안 검사에 크기에 따라 단계 VI 난자를 분리하고 미숙 (작은) 난자를 폐기 : 단계 VI 난자는 동물 반구도 색소 침착과 미성숙 난자보다 큰 약 직경 1.2-1.4 mm이다. </li><li> 20 ° C 형 I - 18에서 난자를 유지주입하기 전에 N OCM. 주 : 아가 로스 코팅 페트리 접시는 접시 난자의 점착을 최소화하기 위해 사용될 수있다. </li></ol> 도서관 성적 증명서 2. 사출 <ol><li> 개발의 적절한 단계 (예를 들어, 신경 판 단계 14 10)에서 추출 방향 cDNA 라이브러리 (15) 사용하여 RNA를 준비합니다. cDNA 라이브러리를 구입하거나 아래의 4 단계 개요 당 상용 키트를 사용하여 하나를 구축합니다. <ol><li> 폴리 (A) + RNA (재료의 표 참조) 풍부하게 제품을 사용하고 제조업체의 지침에 따라 약 5 μg의 mRNA의를 격리합니다. 주 : 사체 (원하는 조직의 미세 절제 다음과 같은 신경 판으로) 유도 조직에 한정 될 수있다 cDNA 라이브러리를 생성하기 위하여 사용되는, 또는 특정 단계에서 전체 배아 일 수있다. </li><li> 제조업체의 지침에 따라, 상용 키트의 cDNA를 생성합니다. </li><li> vecto로 cDNA를 NG 약 20 결찰R은 상용 키트 또는 다른 적절한 벡터에 포함. 적절한 플라스미드 벡터는 mRNA의 안정성 등 5 &#39;β-글로빈 3 폴리 (A) 시퀀스 (PCS2 +, pTnT, pCS105)에 대한 시퀀스를 포함한다. </li><li> 열충격 형질 전환 공급자의 권장 프로토콜을 사용하여 능력 균체 내로 결찰 변환. 참고 : 또는 오픈 리딩 프레임 (ORFeome) 클론의 수집이 시판되고 주입 전 사체를 생성하기 위해 사용될 수있다. </li></ol></li><li> 10 4 복잡성 (10월 4일에서 10월 5일까지 총 복잡성) 10 (3)의 플라스미드 (10) 풀을 준비합니다. 10,000 식민지 각 - 이것은 1000 10 판을 나타냅니다. <ol><li> 37 ℃에서 18 시간, 7 ㎖의 (LB)에 유리 스프레더와 부드러운 압력에 의해 각에서 식민지를 수집 - 플레이트 도서관 문화 10 15-CM LB-암피실린 플레이트 상에, (12)을 성장 </li><li> 0.5 ml의에서 글리세롤 주식을 준비합니다 (-20에서 0.2 ml의 멸균 글리세롤 및 저장에 추가# 730; C), 및 표준 제조사의 지침에 따라 시판중인 DNA 미니 프렙 키트 DNA를 제조 6.5 mL의 나머지를 사용한다. </li></ol></li><li> 1.5 시간 - 효소 (1) 37 ℃에서 16 다이제스트 적절한 제한과 - (2.0 μg의 1.0) 풀링 된 플라스미드 DNA를 선형화. 사용 된 RNA 폴리머 라제 키트의 사양에 물 및 에탄올 침전에 재 부유 한 다음 페놀 / 클로로포름 추출과 선형화 된 DNA를 분리. 제조업체의 지침에 따라 상업 RNA 중합 효소 키트 센스 RNA를 합성. </li><li> 약 20 μm의 직경 (유리 모세관 튜브와 바늘 풀러를 사용하여) 미세 주입 용 바늘을 준비; 교정 된 안구 마이크로 미터와 복합 현미경에 바늘 끝을 측정한다. 참고 : 니들 풀러 설정이 미세 집게로 깨진 경우 원하는 팁 크기를 얻을 수 있도록 좋은 팁 바늘을 생산하는 경험적으로 결정되어야한다. </li><li> (에 점토를 밀어를 준비접시의 바닥)에 균일 한 층 점토 늘어선 35 X 10mm 미세 주입하는 동안 장소에 난자를 유지하는 병렬 홈으로 배양 접시를; 불꽃의 끝에서 융합 쇼핑몰 프로브 또는 파스퇴르 피펫으로 홈을 생산하고 있습니다. 점토에 대한 대안으로서, 엘라스토머 (17)와 톱니 형을 아가로 요리를 준비한다. 점토 늘어선 요리의 행에 1X MBS의 3 % 피콜 (약 2 ㎖)에 넓은 구멍 피펫으로 전송 난자. </li><li> 마이크로 인젝터를 사용하여, 1 NG / NL RNA와 바늘을 작성하고 (난자 세포질의 그리기 방지하기 위해) 약간의 긍정적 인 압력을 생산하기 위해 균형을 조정합니다. </li><li> 적도 지역에서 약 20 NL RNA와 난 모세포를 주입한다. 다음, 피콜-MBS에 남아 1X MBS에 부드럽게 전송 주입 난 모세포에 대한 1 시간을 허용합니다. 이전에 동물 캡 분석에 20 ℃에서 24 시간 - 8 품어. </li></ol> 3. 동물 모자 분석 <ol><li> 미세 집게, 헤어 루프, 곡선 coverslip에 조각, 점토 늘어선 DIS 3 / 4 배 NAM을 준비하고 획득컵 모양의 톱니와 HES는 난자를 개최합니다. 곡선 부분을 생산, (4 MM - - 2 MM × 2 약 1)과 가장자리 광택과 처지까지 불꽃을 통과하는 작은 조각으로 커버 글라스를 따로 끊어서 곡선 coverslip에 조각을 확인합니다. </li><li> X를 기름지게 laevis의 달걀 체외 또는 자연 교배와 단계 낭배하는 문화를 통해 18 - 이전 단계에 의해 정렬에 0.1X MBS (10 실온에서 11 시간 포스트 수정을); 10 배아 - 중간 낭배 (11.5 단계 11)를 수집합니다. </li><li> 3 / 4 배 NAM와 약 반 전체 배양 접시로 전송 배아. 미세 집게 두 쌍을 사용하여, gastrulae에서 수정 (난황) 막을 제거합니다. </li><li> 전송 점토 늘어선 요리 3 / 4 배 NAM (약 2 ㎖)에 난자 홈이 상기 된 개별 배아를 수용하기 위해 인상을 생산, 점토의 개별 노출에 고정. </li><li> 점토 늘어선 요리로 전송 배아 및 G 오프 동물 모자를 잘라astrulae 잘 포셉 두 쌍을 사용하여. 만 아니라 적도 조직 (18) 동물 모자 외배엽을 분리주의하십시오. </li><li> 난자와 접촉하는 동물 캡의 내부 표면과 각 난자의 동물 반구에 동물 캡을 놓습니다. 8 시간 이상 - 6 난자의 표면에 노출 된 내부 층으로 열려있다 (커버 슬립 연락처 동물성 캡 점토 외배엽 평탄화, 곡면 유리 커버 슬립 단편을인가하고, 유리로 하향 압력을인가하여 재조합을 함께 보유 ). , 또는 위쪽을 향 내면에 점토 들여 쓰기에 동물 캡을 배치하고 뚜껑에 난자를 배치; 참고 : 점토의 작은 확장자를 가진 재조합을 고정합니다. </li><li> 20 ° C에서 문화 제어 배아는 분석 무대를 원하는에 도달 할 때까지. </li><li> 커버 슬립 / 진흙을 제거하고 집게와 헤어 루프 외배엽을 분리하여 별도의 재조합. </li><li> MEMFA에서 1 시간 (3.8 %를 외배엽 조각을 수정(MEM) [0.1 M MOPS의 pH가 7.4, 2 mM의 EGTA, 1 mM의 황산 4]에서 포름 알데히드). </li><li> -20 ℃에서 에탄올과 저장소로 MEMFA에서 전송 조각 (및 제어 배아). </li></ol> 에서의 현장 하이브리드 화에 의한 외배엽에서 응답 4. 분석 (ISH) <ol><li> ISH을위한 솔루션을 준비합니다. <ol><li> 1X PBS를 준비 : 0.01 M 인산 완충 식염수, 염화나트륨 0.138 M, pH가 7.4 </li><li> 제조 PBS-트윈 (PTW) 1X PBS, 0.1 % 트윈 -20 </li><li> 100 × 덴 하르트의 용액을 제조 하였다 : 2 % BSA, 2 % 폴리 비닐 피 롤리 돈을, 2 % 피콜 </li><li> 혼성화 완충액을 제조 50 % 포름 아미드, 5 배 SSC, 1 mg의 / ㎖ torula 효모 RNA, 1 μg의은 / ㎖ 헤파린 1X 덴 하르트의 용액, 0.1 % 트윈 -20, 0.1 % CHAPS, 10 mM의 EDTA, DEPC-H 2 O </li><li> 제조 말레 산 완충액 (MAB) : 100 mM의 말레 산, 150 mM의 염화나트륨, pH가 7.5 </li><li> (용해 60 ℃에 열) MAB, 2 % 시약을 차단 : MAB + 블록을 준비 </li><li> 100 mM 트리스 : 알카라인 포스파타제 (AP) 버퍼를 준비합니다pH가 9.5의 50 mM의 MgCl 2, 100 mM의 염화나트륨, 0.1 % 트윈, DH 2 O </li></ol></li><li> RNA 프로브를 준비합니다. <ol><li> 1.5 ML 튜브 (50 μL 반응)에 추가, 상업 RNA 중합 효소 키트를 사용하여 혼합 - NTP의 발굴 : 25.5 μL의 DEPC-H 2 O, 10 μL 5 배 전사 버퍼, 2.5 μL 10 배 발굴-NTP 믹스, 5 μL 100 밀리미터 DTT, 2 μL RNAsin, 2 μL 선형화 DNA 주형 (~ 1 μg의 / μL), 3 μL RNA 중합 효소와 90 분 동안 37 ℃를 품어. </li><li> 2 μL RNA 중합 효소를 추가하고 60 분 동안 37 ℃를 품어. </li><li> 1 % 아가 로스 젤에 반응 2 μL를 확인합니다. </li><li> 1 μL의 RQ1 RNA 분해 효소가없는 DNase를 추가하고 20 분 동안 37 ℃를 품어. </li><li> 50 μL의 DEPC-H 2 O, 25 ㎕의 10 M 암모늄 아세테이트 및 에탄올 313 μL를 첨가하여 침전물을 프로브. -20 ℃에서 보관 하룻밤 (O / N)은 다음 20 분 동안 13,800 XG에서 원심 분리하여 RNA를 복구 할 수 있습니다. 간단히 회전, 500 μL 75 % 에탄올로 세척, 제거에탄올과 공기 건조에 펠렛을 할 수 있습니다. 추가 50 μL의 DEPC-H 2 O </li><li> 0.5 μg의 / μL ~의 최종 농도 하이브리드 버퍼를 추가합니다. </li></ol></li><li> 하이브리드를 위해 조직을 준비합니다. 특별히 언급하지 않는 한, 각 솔루션의 변화를 설명 (약 4 mL)로 정상에 튜브를 입력합니다. <ol><li> 수평 로커에, 10 분 각 75 % 에탄올 / PTW, 다음 50 % 에탄올 / PTW에 튜브 및 이식란으로부터 에탄올을 제거합니다. </li><li> 5 분 로커에 각각 PTW에 세 번 씻으십시오. </li><li> 10 μg의 PTW에서 / μL 테 K 처리로 이동; 바위 튜브를 수직으로 15 분. </li><li> 수직 바위 튜브 - 0.1 M 트리에탄올 아민 pH가 7.8에 두 번 10 분 각 씻어. </li><li> 튜브에 12.5 μL에게 무수 초산을 추가하고 수직으로 5 분 바위. 5 분 동안 추가로 12.5 μL 아세트산 무수물로 반복한다. </li><li> 로커에 분 수직 PTW 5 씻으십시오. </li><li> 로커에 4 % 파라 포름 알데히드 20 분에 REFIX. 열 하이브리드 버퍼60 ℃에. </li><li> 5 분 로커에 각각 PTW에 세 번 씻으십시오. </li><li> 250 μL HYB 버퍼를 각 튜브에서 PTW의 모든하지만 ~ 1 ml의를 제거하고 추가; 부드럽게 섞어 튜브를 소용돌이 친다. 락 튜브를 수직으로 5 분. </li><li> 60 ℃ HYB 버퍼 (0.5 ml)로 교체하고 60 ℃ 10 분에서 부드럽게 교반. 신선한 HYB 버퍼로 교체하고 60 ℃에서 2 4 시간에서 교반. </li><li> 열 프로브 (60) C (3 분)에 (0.5 μg의 HYB 버퍼에 / μL에서 1 ㎖). HYB 버퍼를 제거하고 튜브에 프로브를 추가 할 수 있습니다. O / N은 ​​60 ℃에서 부드럽게 선동. </li></ol></li><li> 항체를위한 조직을 준비합니다. <ol><li> 60 ℃로 따뜻한 HYB 버퍼 및 배 SSC + 0.1 % 트윈 20 솔루션을 제공합니다. </li><li> (- 배 재사용이 -20 ℃에서 프로브를 저장) HYB 버퍼와 프로브 솔루션을 교체합니다. 10 분 동안 60 ℃에서 씻으십시오. </li><li> 배 SSC-트윈에서 60 ℃ (20 분 교반과 함께 각)에서 세 번 씻으십시오. </li><li> 0.2X SSC-트윈에서 60 ℃ (20 분 교반과 함께 각)에서 세 번 씻으십시오. </li><li> 로커에 15 분마다 수평에 대해 MAB (RT)에 두 번 씻으십시오. </li><li> 1 ML의 MAB + 블록을 추가합니다. 로커에 수직으로 2 시간을 씻으십시오. </li><li> 1 / 2,000 안티 digoxygenin-AP를 포함하는 1 ML의 MAB + 블록에 전송. 4 주 / N에서 수직 바위. </li></ol></li><li> 색상 시약을위한 조직을 준비합니다. <ol><li> MAB와 항체 솔루션을 교체; MAB 5 분마다 수평 로커에 세 번 씻는다. </li><li> MAB 1 시간에 각각의 수평 로커에 세 번 씻으십시오. </li><li> AP 버퍼 10 분에 각각 수평 로커에 두 번 씻으십시오. </li><li> 여러 웰 플라스틱 트레이로 조직을 이동 AP 버퍼를 제거하고 BM에게 보라색 시약을 추가 (1 ~ ㎖). 반응이 O / N 어두운 5 분에 할 수있게합니다. </li><li> 적절한 염색이 달성되면, PTW의 바이알에 조직을 전송. 로커에 PTW에 두 번 10 분 각각을 씻으십시오. </li><li> 로커에 4 주 / N에서 Bouin의 솔루션에 고정합니다. </li><li> 실온에서 Bouin의 세 70 % 에탄올 / 30 % PTW 세척과를 세척 할 것. T를 진행필요한 경우 18 에피 조명 및 현미경에 장착 된 카메라, 표백을 사용 O 이미지 캡처. </li></ol></li></ol> 5. SIB 선정 및 복제 <ol><li> 가장 높은 활성을 선택 풀 (외배엽 조직에 가장 큰 응답). </li><li> (위의 프로토콜 섹션 2를 사용) 이전 단계의 식민지를 가장 높은 활성을 가진 풀의 글리세롤 주식을 (적절한 밀도 LB로 희석) 약 십분의 일에 열 새로운 판을 판 적정한다. </li><li> 활동이 하나의 식민지로 추적 될 때까지 풀의 크기를 줄입니다. </li><li> 벡터 표준 프라이머를 이용하여 DNA 서열을 얻습니다. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

유능한 외배엽에서 반응을 유도 할 수있는 유전자의 기능적 클로닝 여기 기재된 방법은 유전자 산물의 넓은 범위를 식별하기 위해 사용될 수있다. 이 방법은 표현의 복제 기술과 조직 유도 분석을 결합하여 과거의 일에 확장합니다. 우리는 RNA 주입 다음, 직접 또는 간접적으로 유도 인자의 생성 소스로서 제노 푸스 난 모세포의 대사 경로를 이용한다. 이것은, 관심 cDNA 라이브러리 또는 클?...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

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			<td style="width:233px;">cells for transformation of cDNA library</td>
		</tr>
	</tbody>
</table>

functional cloning using a xenopus oocyte expression system

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

동물 캡 조직 응답, 난자에 주입의 mRNA의 발현에 대응하여 현장 하이브리드 (그림 2, 표 1)에 의해의 Otx2의 발현을 측정 하였다;의 Otx2이 렌즈 placode를 통해 신경관 폐쇄에서 추정 렌즈 외배엽 (PLE)로 표현된다 농축 (19). 도의 Otx2 PLE 외부 전방 신경 외배엽뿐만 아니라 비 - 신경 외배엽 헤드 표현이기 때문에,이를 신경과 p...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

를 사용하여 기능성 복제<em&gt; Xenopus의</em&gt; 난자 발현 시스템

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

functional-cloning-using-a-xenopus-oocyte-expression-system

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

8.0K 조회수.  Shepherd University. We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

비디오: Functional Cloning Using a Xenopus Oocyte Expression System

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

이해 간체를 사용하여 초기 형성기<em&gt; 현장에서</em에서&gt; 하이브리드 프로토콜<em&gt; 아프리카 발톱 개구리</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

연구

발생학

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

애벌레 Zebrafish의 피부에 유도 GAL4 / UAS 식 시스템을 사용하여 선천성 면역과 종양 전 세포 상호 작용의 라이브 영상

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

동축 노즐을 사용하여 다 능성 줄기 세포의 알긴산 캡슐화

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

공 촛점 분석을 사용하여<em&gt; 아프리카 발톱 개구리 laevis의</em&gt;의 Wnt 및 쉬 Morphogen 그라디언트의 변조기를 조사하기 위해

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Photoconvertible PSmOrange 시스템을 사용하여 GFP 유전자 변형 Zebrafish의 세포를 추적

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

개발에 미세 주입을 대상으로 기술<em&gt; Xenopus의</em&gt; 신장

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

자세한 공간 식지도에서 동적 식 데이터의 시간적 순서

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

사용 모성 유전자 제품의 기능 조작<em&gt; 체외</em제브라 피쉬&gt; 난자의 성숙

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

태아 마우스 심장 혈관 이미징 높은 주파수 초음파 (30/45 m h Z) 시스템을 사용 하 여

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

내피 콜로니 형성 세포및 중간엽 줄기 세포에 의해 형성된 공동 배양 스페로이드를 이용한 3차원 혈관신생 분석 시스템

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

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애벌레 Zebrafish의 피부에 유도 GAL4 / UAS 식 시스템을 사용하여 선천성 면역과 종양 전 세포 상호 작용의 라이브 영상

동축 노즐을 사용하여 다 능성 줄기 세포의 알긴산 캡슐화

공 촛점 분석을 사용하여

Photoconvertible PSmOrange 시스템을 사용하여 GFP 유전자 변형 Zebrafish의 세포를 추적

개발에 미세 주입을 대상으로 기술

자세한 공간 식지도에서 동적 식 데이터의 시간적 순서

사용 모성 유전자 제품의 기능 조작

태아 마우스 심장 혈관 이미징 높은 주파수 초음파 (30/45 m h Z) 시스템을 사용 하 여

내피 콜로니 형성 세포및 중간엽 줄기 세포에 의해 형성된 공동 배양 스페로이드를 이용한 3차원 혈관신생 분석 시스템