이 절차는 이식 기술은 빌 제프리의 실험실에 의해 동굴 물고기를 위해 개발된 광범위하게 따릅니다.

1 부 : 배아 준비 이 프로토콜에서는, 우리는 가상 zebrafish 렌즈 돌연변이 거하고 있음을 나타내는 JJ XY 기호를 사용합니다. <ol><li> Zebrafish의 변종은 14h light/10h 어두운주기 28.5 C에서 표준 생선 시설 조건에서 유지됩니다. </li><li> 저녁에는 자리 남성과 zebrafish 변형 JJ XY의 여성 : AB / TU, 서로를 구분하는 구분선과 탱크에 TG (mGFP). 이 십자가의 자손은 모든 조직에서 GFP의 transgene을 표현되며, 기부자로 사용됩니다. 병렬로, 비 - 유전자 변형 야생 형 동물 사이의 경계를 설정하는 동일한 절차를 사용합니다. 본 실험의 목적에 따라, 한 기증자 또는 호스트로 JJ XY의 로커스에서 야생 타입 동물을 교차 수 있습니다. </li><li> 빛이 아침에 켜지 후 한 시간 이내, 친구에게 물고기를 허용하도록 디바이더를 제거합니다. </li><li> 15-30분 100 X 15mm 배양 접시에 배아를 수집 후, 그들을 청소하고, 매체 (계란 물)을 변경합니다. </li><li> 원하는 시간까지 28.5 C 배양기에서 배아 보관하십시오. </li><li> 언제 이식 준비, 날카로운 집게 두 쌍이있는 수동 chorion을 제거하고 30 분 동안 칼슘 무료 ZFR에서 0.2 % EDTA (에틸렌 다이아 민 테트라 초산)의 배아를 품어. </li></ol> 2 부 : 절개의 바늘의 작성 <ol><li> 및 기증 렌즈를 이동하고 호스트에 삽입하는 데 사용하는 무딘 바늘, 렌즈 주위 조직을 잘라 chorion을 제거하고, 아가로 오스의 릴리스 배아하는 데 사용됩니다 날카롭게 한 : 이식 절차는 바늘의 두 가지 유형이 필요합니다. 여기이 바늘을 만드는 방법을 보여줍니다. </li><li> 첫째, 파스퇴르 피펫의 끝은 다이아몬드 칼을 이용하여 차단해야합니다. 다음 길이의 약 절반 안에되도록 파스퇴르 피펫 팁으로 모세관 유리를 삽입합니다. 이것은 쉽게 다음 단계에서 유리에 그것을 용융하여 텅스텐 와이어를 고정하는 것입니다. </li><li> 다음 모세관의 열린 끝을로 얇은 텅스텐 와이어를 삽입합니다. 알콜 램프로 철사 위에 유리를 용융. 손으로 바늘의 다른 쪽 끝을 비틀면서 집게를 사용하여 금속 와이어와 함께 꾸준한 유리 끝을시오. 부드럽게 유리는 금속 주위에 나선 자리에서 재밌해야합니다. </li><li> 당신이 뭉툭한 팁 바늘하고있다면이 절차의 마지막입니다. 날카롭게 바늘을 만들려면, 아주 좋은 팁이 생성되도록 금속을 굽기, 1-1.5 분 알콜 램프를 통해 텅스텐 와이어를 잡아. microscropy.It에 의해 팁의 품질은 이식 직전 몇 초 동안 불꽃에 두 바늘을 소독하는 것이 좋습니다 확인하십시오. </li></ol> 파트 3 : 시약의 준비 <ol><li> 칼슘 무료 Zebrafish 링거스 (ZFR) 솔루션 (116 MM NaCl, 2.9 KCl MM, 5 MM HEPES, pH를 7.2) </li><li> 칼슘 무료 Zebrafish 링어의 솔루션 (산도 7.2)에서 0.2 % EDTA (에틸렌 다이아 민 테트라 초산) </li><li> 칼슘 무료 ZFR에서 1.2 % 아가로 오스 (낮은 융점) </li><li> 칼슘 무료 ZFR에서 0.2 % 아가로 오스 (낮은 융점) </li><li> 리터 당 배아 매체 (산도 7.0이 포함되어 있습니다 : 10 ML 행크스 솔루션 # 1, 1ml 행크스 솔루션 # 2, 10ml 행크스 솔루션 # 4, 10ml 행크스 솔루션 # 5, 0.35 g의 나트륨 중탄산염, 2M 300 UL을 HCL, 페니실린 - 스트렙토 마이신 500,000 Zebrafish 도서 (오레곤 프레스 대학, 2000)에서와 마찬가지로 U). </li><li> 텅스텐 와이어, 도금, 0.1mm 직경 알파 Aesar </li><li> 모세관, 1.0mm 직경, 세계 정밀 계측기 </li></ol> 4 부 : 이식 절차 <ol><li> 플라스틱 원심 분리기 튜브에서 물 목욕 40 C에 preheated 칼슘 무료 ZFR에서 1.2 % 낮은 용융 점 아가로 오스를 준비합니다. </li><li> 원하는 단계 (30 HPF, 예를 들면)에서, 파스퇴르 피펫으로 배아를 타고 천천히 원심 튜브에 그들을 추방하고, 서서히 원심 튜브에 그들을 추방하고 약 5 초 동안 품어. 기증자와 호스트는 별도 incubated해야합니다. </li><li> 피펫으로 원심 튜브에서 1.2 % 아가로 오스의 배아를 가지고 100mm 폴리스티렌 페트리 접시 두 평행 행에서 그들을 마련, 호스트와 기부자는 별도의 유지. 천천히 그리고 신중하게 그들을 pipetting하여, 대부분의 태아는 아가로 오스에서 양쪽에 누워 것입니다. 당신은 아가로 오스가 굳은 전에 안구가 직면되도록 무딘 바늘을 사용하여이 위치에 거짓말하지 않는 사람을 방향해야합니다. </li><li> 응고에 1.2 % 아가로 오스 기다립니다, 그리고 0.2 % 낮은 융점 아가로 오스 솔루션을 오버레이. </li><li> 날카롭게 바늘을 사용하면, 눈 주위에 아가로 오스를 제거, 그리고 아주 조심스럽게 작은 스트로크를 사용하여 호스트 및 기증 배아에서 렌즈를 잘라. 그럼으로 그것을 찢어하지 않는 렌즈에 그냥 가까운 정도 잘라 있는지 확인뿐만 아니라, 그래서 당신은 호스트 눈의 나머지 부분에서 너무 많은 조직을 제거하지 마십시오. </li><li> 일단 무료로 렌즈가 중간에 떠 있습니다. 무딘 바늘을 사용하여 조심스럽게 기증자를 밀어렌즈는 단지 호스트 렌즈가 정상적으로 될 어디의 위치 이상에 다음 무딘 바늘로 눈 안으로 그것을 아래로 밀어. 호스트 렌즈가 삭제됩니다. </li><li> 30~60분에 대한 1.2 % 아가로 오스의 기증자와 호스트 배아를 남겨 다음 날카로운 바늘을 사용하여 아가로 오스에서 그들을 출시하고, 배아 매체와 함께 24 잘 판에 그들을 전송합니다. 각각의 배아는 별도의 우물을 점유한다. 제대로 때문에 호스트가 어떤 기증자에 해당하는 취소되는 우물 레이블을해야합니다. </li><li> 기증자 파생 렌즈 GFP 형광에 따라 같은 동물의 unoperated 측면에 호스트 렌즈에서 구분할 수 28.5 C.에서 알을 품다. 섹션은 또한 렌즈의 크기를 측정하고 제대로 차별화 여부를 확인 할 수 있습니다. </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="그림 1" /> 그림. 렌즈 이식에 사용 날카롭게 바늘 1 낮은 배율 이미지. 모세관 유리 (B)은 파스퇴르 피펫 (A)에 삽입하고, 얇은 텅스텐 와이어 (C)은 모세관의 열린 끝을 삽입됩니다. 와이어를 통해 유리는 알콜 램프로 부드럽게합니다. 손으로 파스퇴르 피펫의 다른 쪽 끝을 비틀면서 집게를 사용하여 금속 와이어와 함께 꾸준한 유리 끝을시오. 부드럽게 유리 장소에 그것을 근사한, 금속 주위에 나선 것입니다. <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> 그림. 렌즈 이식에 사용되는 바늘의 이가지 2 팁. <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="그림 3" /> 그림. 3 태아는 30hpf에서 렌즈 이식을 받았습니다. 표시는 기증자의 배아 (A, B), 이식 렌즈 (C, D)으로 호스트 눈, 및 호스트 배아의 제어 측면 (E, F)입니다. 표시된 각 눈은 전송 빛과 자외선 조명 모두에 표시됩니다. 사진은 48 HPF에서 찍은. Q01 [9] 유전자 변형 라인이 실험에서 사용되었습니다.

<ol>
	<li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens.">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev Biol. 231, 63-76 (2001).</li><li>Yamamoto, Y., Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+for+the+lens+in+cave+fish+eye+degeneration.">Central role for the lens in cave fish eye degeneration.</a> Science. 289, 631-633 (2000).</li><li>Easter, S. S., Nicola, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+vision+in+the+zebrafish(Danio+rerio).">The development of vision in the zebrafish(Danio rerio).</a> Dev Biol. 180, 646-663 (1996).</li><li>Schmitt, E., Dowling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+eye+morphogenesis+in+the+zebrafish.+Brachydanio+rerio.">Early eye morphogenesis in the zebrafish. Brachydanio rerio.</a> J.comp. Neuro. 344 (4), 532-542 (1994).</li><li>Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+forward+genetics--analysis+of+neuronal+diversity+and+patterning+in+the+zebrafish+retina.">Harnessing the power of forward genetics--analysis of neuronal diversity and patterning in the zebrafish retina.</a> Trends Neurosci. 23 (11), 531-541 (2000).</li><li>Malicki, J., Pujic, Z., Thisse, C., Thisse, B., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+to+the+analysis+of+eye+development+in+zebrafish.">Forward and reverse genetic approaches to the analysis of eye development in zebrafish.</a> Vision Res. 42 (4), 527-533 (2002).</li><li>Avanesov, A., Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+study+neurogenesis+in+the+zebrafish+retina.">Approaches to study neurogenesis in the zebrafish retina.</a> Methods Cell Biol. 76, 333-384 (2004).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutagenesis+yields+eye+morphological+mutants+with+retinal+and+lens+defects.">Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.</a> Vision Res. 42, 535-540 (2002).</li><li>Godinho, L., Mumm, J. S., Williams, P. R., Schroeter, E. H., Koerber, A., Park, S. W., Leach, S. D., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+amacrine+cell+neurites+to+appropriate+synaptic+laminae+in+the+developing+zebrafish+retina.">Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina.</a> Development. Nov 132, 5069-5079 (2005).</li></ol>

여러 단계 렌즈 transplantations 중 특별한주의가 필요합니다. <ol><li> 바늘을 날카롭게 : 그것은 렌즈 이식을 수행하기 전에 몇 가지 바늘을 준비하는 것이 좋습니다, 그리고 바늘 끝이 가능한 얇은해야합니다. 두꺼운 바늘은 치료 눈에 실패를 초래하기 때문에 넓은 직경이 더 많은 조직을 찢어 것입니다. </li><li> 배아를 정리 : 그들의 측면에 누워되도록 마우스 이식하기 전에 배아를 위치합니다. ?...

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lens transplantation in zebrafish and its application in the analysis of eye mutants

렌즈는 광학 컵 [1,2]의 개발에 중요한 역할을합니다. 모델 유기체로 zebrafish를 사용하여 렌즈 개발에 관한 질문은 해결할 수 있습니다. zebrafish 작은 크기, 높은 생산력, 짧은 수명주기, 그리고 관리의 용이성 등 여러 가지 유리한 특성에 의한 유전자 연구에 유용합니다. 렌즈 개발 zebrafish에 급속하게 발생합니다. 72 HPF으로 zebrafish 렌즈 기능 성숙이다 [3]. 풍부한 유전 및 분자 자원 zebrafish 연구를 지원하기 위해 사용할 수 있습니다. 또한, 다른 척추 동물들에게 zebrafish 눈의 유사성은 인간의 결함이 뛰어난 동물 모델 [4-7]과 같이 사용에 대한 기초를 제공합니다. 여러 zebrafish의 돌연변이가 어떤 경우에는 렌즈 조직의 완전한 변성에 이르게 세포 사망의 높은 수준 [8]. 포함한 렌즈 이상 전시 렌즈 이상은 본질적인 원인이나 주변 조직과 결함이 상호 작용에 의한인지 확인하려면, 야생 형 안구에 돌연변이 렌즈의 이식이 수행됩니다. 를 사용하여 화재를 광택 금속 바늘, 돌연변이 또는 야생 형 렌즈는 신중하게 기증 동물의 해부를하고, 호스트로 전송됩니다. 야생 타입과 돌연변이 조직을 구분하기 위해 유전자 변형 라인은 기증자로 사용됩니다. 이 줄은 렌즈를 포함한 모든 조직의 막 - 바운드 GFP를 표현합니다. 이 이식 기술은 zebrafish 렌즈 돌연변이의 연구에 필수적인 도구입니다.

Watch this Scientific Journal Video about Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants at JoVE.com

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

렌즈 개발은 다른 조직과 상호 작용을 포함한다. 여러 zebrafish 아이 돌연변이 비정상적으로 작은 렌즈의 크기가 특징입니다. 여기 우리는이 표현형은 본질적 원인이나 렌즈를 둘러싸고있는 조직과 결함이 상호 작용에 의한 있는지 여부를 확인하기 위해 렌즈 이식 실험을 보여줍니다.

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 ...

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JoVE Journal

Biology

11.0K 조회수.  The Second Teaching Hospital of Jilin University. 렌즈 개발은 다른 조직과 상호 작용을 포함한다. 여러 zebrafish 아이 돌연변이 비정상적으로 작은 렌즈의 크기가 특징입니다. 여기 우리는이 표현형은 본질적 원인이나 렌즈를 둘러싸고있는 조직과 결함이 상호 작용에 의한 있는지 여부를 확인하기 위해 렌즈 이식 실험을 보여줍니다.

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

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 ...

연구

생물학

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

터미널 차별 유학을위한 준비 및 랫 렌즈 상피 Explants의 문화

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

차 문화와 Corti의 Murine 오르간의 플라스미드 Electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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 ...

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 ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

드로소필라의 온도 선호도 행동과 Circadian 리듬의 설계 및 분석

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

성인 Zebrafish의 신장에서 네프론의 구성 및 기능 분석

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

마우스 렌즈의 압축 강성을 평가하는 유리 Coverslips는 순차적으로 응용 프로그램 : 스트레인과 형태 학적 분석

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

총회와 &#39;전단 반지&#39;의 응용 프로그램 : 궤도, 단방향 및 정기 유체 흐름 및 전단 응력에 대한 소설 내피 모델

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...