우리는 suturing와 도움 EC 리아 감사하고, R. 에디어 및 zebrafish 보험 L. Gyr. AJD는 하버드 줄기 세포 연구소, 신장학의 미국 사회, 그리고 NIH / NIDDK (P50DK074030)에 의해 지원되었다. CQD는 의학적 발견에 대해 매사 추세츠 종합 병원 - 기금에 의해 지원되었다.

1. 비 - 유전자 변형받는 물고기의 컨디셔닝. <ol><li> 이식에 필요한 시스템에서 가장 큰 남성 생선을 선택합니다. </li><li> 약 3 분 또는 수영 있지만, 심장 박동 중지 너무 오래하지 멈출 때까지 0.02 % Tricaine에있는 물고기 (산도 7) 장소. </li><li> 작업대 위에 젖은 종이 타월에 anesthetized 생선을 넣어. </li><li> 물고기의 intraperitoneal 구멍에 물을 20 UL에서 gentamicin 40 UG를 삽입하는 인슐린 주사 바늘을 사용합니다. 신장 재생 연구의 경우,이 단계는 향상된 engraftment을위한 유리한 환경을 유도하는 것이 중요합니다. </li><li> 복구를 위해 시스템 물로 다시 생선을 넣어. </li><li> 물고기 다루기 및 진동 등의 외부 자극에 민감 수 있습니다. 복구 3 시간 후, 감마 방사선 조사 25 회색으로 생선을 취급. </li><li> 이전 이식 음식없이 3 일간 다시 시스템에 물고기를 넣어. </li></ol> 2. 전구 기증자 세포의 준비는 유전 형광 마커 (EGFP 또는 mCherry)로 표시. <ol><li> 그냥 이전에 수술로 이식하는 기증자 세포를 준비하고 얼음에 있습니다. </li><li> 신장과 조혈 전구 세포를 포함하는 유전자 변형 라인 : 신장 연구, 우리는 TG (EGFP cdh17)에서 전체 신장 골수 세포를 사용합니다. 이러한 전구 세포는 유전자 표시하고 그들이 합치다 및 신장 상피 세포로 차별화하는 경우에만 EGFP을 표현합니다. angioblast의 progenitors 이식을 위해 : 조혈 줄기 세포 또는 TG (EGFP fli1a)에 : 표시 전구 세포의 다른 종류는 또한 TG (EGFP SCL)으로 사용할 수 있습니다. </li><li> 5 분 0.2 % Tricaine에서 그들을 배치로 3 성인 유전자 변형 물고기를 안락사. </li><li> 복부 측면을 따라 정중선 절개를 만들기 위해 머리와 꼬리, 그리고 해부 가위를 제거하는 소독 면도날을 사용합니다. </li><li> 입체경에서 족집게로 수영 방광을 포함하여 뱃속에있는 내장을 모두 제거합니다. 구멍의 지느러미 벽에 부착된 평면과 색소 오르간있는 신장을 제거하지 않도록주의하십시오. </li><li> 다음, 등 부분의 벽면에서 신장을 해부하다하고 2퍼센트 소태아 혈청 (1X PBS / 2% FCS)와 보충 차가운 인산염 버퍼 호수 300 UL을 포함한 1.5 ML의 원심 관에 넣습니다. </li><li> 그것이 균질 될 때까지 전단 신장에 유봉을 사용합니다. </li><li> 50 ML 팔콘 튜브 안에 40 음 셀 스트레이너를 삽입하고 느슨한 세포 팔콘 튜브에 통과 필터 수 있도록 셀 스트레이너 위에 셀 솔루션을 전송할 수 있습니다. </li><li> 부드럽게 얼룩 더욱 조직을 깰 수있는 여과기에 남아있는 조직의 큰 조각을하는 유봉을 사용합니다. </li><li> 팰컨 튜브 셀 솔루션을두고 스트레이너의 잔여 세포를 수집하는 1X PBS / 2% FCS 1.2 ML로 스트레이너 씻으십시오. </li><li> ° C 300 RCF에서 5 분 4 새로운 1.5 ML 튜브와 원심 분리기에 셀 솔루션을 전송합니다. </li><li> 1X PBS / 2% FCS 1.5 ML로 세포 펠렛을 씻고 끈적끈적한 세포의 대단히 짧은 시간을 필터링하고 다시 원심 분리기에 새로운 스트레이너를 통해 전달한다. </li><li> 1X PBS의 / 2% FCS 200 UL의 세포 펠렛을 Resuspend 및 PCR 튜브에 전송할 수 있습니다. </li><li> PCR 튜브 (4 ° C, 300 RCF, 1 분)을 원심 분리기 및 뜨는의 대부분을 제거합니다. </li><li> 2-5 UL 최종 볼륨을하고 얼음에 저장하는 잔류 뜨는의 세포를 Resuspend. </li><li> 최종 세포 수율은 신장 약 2,000,000 전지해야합니다. </li><li> hemocytometer로 세포의 수를 계산하고 5 농도를 조절 UL 회 X 10 5 세포. </li></ol> 3. 해밀턴 주사기로 세포 솔루션을로드합니다. <ol><li> 멸균 물 3 회와 해밀턴 주사기 린스. </li><li> 3 회 에탄올 75%과 주사기를 씻어. </li><li> 1X로 다시 PBS / 2퍼센트 FCS 3 번 주사기를 씻어. </li><li> 그런 다음 셀 솔루션 1 UL 방금 전에 주사로 약 5 × 10 5 세포를 포함하고있는 주사기를로드합니다. </li></ol> 4. 직접 신장에 전구 세포 이식. <ol><li> 물고기가 안움직여 약 3 분, 또는 때까지 아니지만 너무 오래 심장 박동 중지에 대해 0.02 % Tricaine에서 에어컨받는 물고기를 마취. </li><li> stereomicroscope에서 이식 측면을 건조하기 위해, 최대 왼쪽 복부 쪽을 향해 머리를 종이 타월에 생선을 믿는다. </li><li> 그런 다음 마른 측면을 노출하기 위해 생선을 통해 롤. </li><li> 멸균 핀셋을 사용하여 아가미에 즉시 후부 지역에서 비늘을 제거합니다. </li><li> 핀셋으로 조직을 안정화하면서 때론 정중선에이 지역에서 1cm 측면 절개를합니다. </li><li> 깊은 충분히 절개를 계속하면서 핀셋을 사용하여 조직을 노출머리 신장을 폭로합니다. </li><li> 신장의 시각화를 방지 출혈이 많이있다면, 물고기를 euthenize 다른받는 물고기와 함께 다시 시작. </li><li> 머리 신장을 노출하기 위해 조직이 열려 캐고 있지만, 직접 머리 신장에 세포 솔루션 1 UL을 주입. </li><li> 조직이 주입된 세포의 누출 방지하기 위해 장소로 다시 떨어지는 것을 그럼, 주사기 바늘이 검색되는 것과 동시에, 족집게를 놓습니다. </li><li> 바늘 드라이버와 절개의 각 측면에있는 근육을 통해 피어스 그것으로 봉합 바늘을 잡아. </li><li> 이 매듭 묶는하고 초과 봉합을 차단. 오직 하나의 봉합은 1cm 절개를 위해 필요합니다. </li><li> 진정 진통 효과와 생선을 제공하는 5 시간 Tricaine의 백만 당 10 부품을 포함하는 물고기 물 속에 물고기를 놓습니다. </li><li> 정상 feedings와 복구 시스템으로 다시 물고기를 놓습니다. </li><li> 회복의 7-18일 후, 우리는 물고기를 해부하다 라이브 EGFP 형광에 의해 신장 전구 세포 engraftment를 분석합니다. </li></ol> 5. 대표 결과 : 이식의 전체적인 전략은 그림 1에서 그림입니다. 우리 신장 연구, 신장이 해부되고 EGFP 형광으로 분석했다. 신장 전구 세포의 Engraftment과 차별화는 7 일 이후 이식 빠르면 감지됩니다. 이것은 EGFP의 여러 클러스터 + 신장 상피 세포 (그림 2)의 존재에 의해 분명하다. 십팔일 게시물 이식함으로써, 우리는 기증자의 세포 (그림 3) 신장 이식 조직을 형성한다는 의미, 24 EGFP + nephrons의 평균을 감지. 장기 engraftment을 증명하기 위해, 우리는받는 물고기 59일 게시물 이식 (그림 4)를 해부 했어요. 이 물고기는 신장 전구 세포가 철새 것을 제안, 먼 사이트 (흰색 화살촉)에서 여러 nephrons 이외에 주입의 사이트 (빨간 화살촉)에서 많은 nephrons했다. 이 기술은 세포 engraftment을 감지까지 약 3 개월이 소요 심장을 통해 혈액 순환에 주입하는 경우에 비해보다 빠르고 효율적입니다. <img src="/files/ftp_upload/2725/2725fig1.jpg" alt="그림 1"/> 그림 1. 이식. 비 유전자 변형받는 물고기의 전체 구조는 이식 세포의 면역 거부를 억제하기 위해 조사하고 있습니다. 사흘 후, 절개는 생선과 기증 전구 세포의 측면에서 이루어집니다은 노출된 머리 신장에 주입하고 있습니다. 절개는 복구를 위해 시스템에 다시 배치되는 하나 봉합와 물고기와 닫힙니다. 받는 물고기의 신장은 나중에 포인트로 해부하고 engraftment 및 시조 활동에 대한 분석입니다. <img src="/files/ftp_upload/2725/2725fig2.jpg" alt="그림 2"/> 그림 2. 7 일 후에 전구 세포 Engraftment. 신장 전구 세포의 Engraftment은 같은 EGFP + 신장 상피 세포의 클러스터의 존재 (- 확대보기 삽입된 페이지)에 의해 결정, 7 일 이후 이식의 머리 신장의 주입 측면에 감지됩니다. <img src="/files/ftp_upload/2725/2725fig3.jpg" alt="그림 3"/> 그림 3. 십팔일 후 전구 세포의 Engraftment 십팔일 게시물 이식 후, 24 EGFP의 평균 + 기증자 파생 nephrons를 (삽입 - 개인 EGFP + nephrons의 확대보기) 감지.. <img src="/files/ftp_upload/2725/2725fig4.jpg" alt="그림 4"/> 그림 4. 59일 후 Engraftment 및 전구 세포의 마이 그 레이션. 우리는 심지어 59일 (레드 애로우 헤드) 후 이식 후 긴 전구 세포 engraftment을 감지 계속. 나중에이 시점에서, 기증자 파생 nephrons은 전구 세포의 이주와 일치하는 주입 (흰색 화살표 머리)의 사이트에서 떨어진 장소에서 감지됩니다.

<ol>
	<li>Hopkins, C., Li, J., Rae, F., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+options+for+kidney+disease.">Stem cell options for kidney disease.</a> The Journal of Pathology. 217, 265-281 (2009).</li><li>Brittijn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+lethal+irradiation+in+zebrafish+and+rescue+by+hematopoietic+cell+transplantation.">Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation.</a> Blood. 104, 1298-1305 (2004).</li></ol>

관심 없음 충돌 선언하지 않습니다.

세포 이식 기술은 재생 연구에 중요한되며 줄기 세포 활동을 측정하는 황금 표준 분석하고 있습니다. 여기서 우리는 줄기 및 전구 세포의 활동을 결정하기 위해 직접 성인 zebrafish의 신장에 세포를 이식하는 방법을보고합니다. 이 절차는 직접 신장에 세포의 주입 다음에 머리 신장을 노출하기 위해 근육을 통해 절개 포함됩니다. 절개는 단일 봉합과 함께 폐쇄하고 운영 물고기 90~100%의 생존 비율을 가지고있다. 우리의 실험에서, engrafted 신장 전구 세포는 7 일 게시 이식에 의해 이식 물고기의 80-100%에서 감지됩니다. 이 기법 중 하나는 한계는 절개 쉽게 메스에 의해 파열 수있는 등의 대동맥, 근처에있다는 것입니다. 그러나, 일단 마스터, 출혈의 속도 미만 %까지 줄일 수 있습니다. 우리의 방법은 신장에 다른 세포 유형 (혈액, stromal, 또는 혈관)뿐만 아니라 작은 분자를 제공하기 위해 적응 수 있습니다. 이 기법의 개발은 사전에 재생 연구를 도움 줄기 및 전구 세포의 잠재력은 생체내에서 평가하는 것을 허용합니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 인슐린 주사기 바늘 (28 게이지) </td><td> 백톤 디킨슨 </td><td> 329461 </td></tr><tr><td> 셀 스트레이너 (40 음) </td><td> 백톤 디킨슨 </td><td> 352340 </td></tr><tr><td> Tricaine </td><td> 시그마 </td><td> A5040 </td></tr><tr><td> 해밀턴 주사기 (26 게이지) </td><td> 시그마 </td><td> 20,734 </td></tr><tr><td> Ethilon 봉합사 (크기 9-0) </td><td> Ethicon </td><td> 2819G </td></tr></tbody></table>

transplantation of cells directly into the kidney of adult zebrafish

손상된 조직에 줄기 또는 조상 세포의 이식에 따라 재생 의학은 만성 질환 1 다양한 치료 가능성이있다. 그러나 대부분의 기관이 구조에 액세스할 수 외과 방법을 개발의 필요성을 necessitating 쉽게 액세스할 수 없습니다. 이 비디오를 문서에서, 우리는 직접 성인 zebrafish, 중생과 질병이 공부를 인기있는 모델의 신장에 세포를 이식하는 방법을 설명합니다. 받는 사람 물고기는 주입 세포 3 면역 거부를 억제하기 위해 방사선에 의해 사전 냉방 있습니다. 우리는 머리 신장이 직접 기관에 세포의 주입 다음에 물고기의 측면에 가로 절개에 의해 노출 수있는 방법을 보여줍니다. 모든 셀 기반의 재생 치료의 황금 표준 - 신장과 조혈 엽 성의 전구 물질의 혼합 인구로 구성된 찬란 분류 전체 신장 골수 세포를 사용하여, 우리는 nephron의 progenitors가 합치다하고 새로운 신장 조직으로 차별화 수 보여줍니다. 이 기법은 신장뿐만 아니라 다른 내부 장기의 줄기 또는 조상 세포 및 / 또는 작은 분자를 정화하고 추가 재생 의학을 공부하기 위해 다양한 모델로 zebrafish를 향상 전달하기 위해 적용할 수 있습니다.

Watch this Scientific Journal Video about Transplantation of Cells Directly into the Kidney of Adult Zebrafish at JoVE.com

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

세포 이식은 조직 재생을 연구와 질병의 세포 기반 치료법​​을 개발하기위한 필수적인 기술입니다. 우리는 여기서 성인 zebrafish 물고기의 신장에 직접 레이블 유전자 세포의 이식을 허용 microsurgical 기법을 보여줍니다.

직접 성인 Zebrafish의 신장에 세포의 이식

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

transplantation-of-cells-directly-into-the-kidney-of-adult-zebrafish

Research

JoVE Journal

Biology

15.0K 조회수.  Massachusetts General Hospital. 세포 이식은 조직 재생을 연구와 질병의 세포 기반 치료법​​을 개발하기위한 필수적인 기술입니다. 우리는 여기서 성인 zebrafish 물고기의 신장에 직접 레이블 유전자 세포의 이식을 허용 microsurgical 기법을 보여줍니다.

비디오: Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

성인 Zebrafish의 전체 신장 마로우의 이식

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

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Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

당뇨병 마우스의 신장 캡슐으로 췌장 아일 레츠의 이식

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

렌즈 Zebrafish의 이식과 눈 돌연변이의 분석에의 응용

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

성인 Zebrafish에서 기관의 해부

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Syngeneic Zebrafish의 제한 희석 세포 이식을 사용하여 종양 - 증식 세포의 주파수 Quantifying

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

성인 Zebrafish의 신장의 해부

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

생성중 성인 Zebrafish 테일 핀으로 Morpholinos의 생체내 Electroporation에

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

성인 Zebrafish의를 Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...