우리는 zebrafish 양육 그녀의 지원을 위해 양 팬 쉔 감사합니다. 이것은 박사 빙 후진타오 (중국 과학 아카데미, &quot;바이 랜 지 후아&quot;NSFC 30870833 중국 국립 기본 과학 프로그램, 2009CB941302)에 fundings에 의해 지원됩니다. 모든 동물 실험은 과학 및 중국 (USTC) 동물 자원 센터와 대학 동물 케어 및 사용위원회의 기술 대학에 의해 명시된 지침과 규정에 따라 실시되었다.

부 I : OKR 장치 구축 자세한 사항은 비디오 데모에 표시됩니다. 부 II : 성인 Zebrafish의 OKR 당신의 실험실 OKR 장치를 구축있다면, 성인 zebrafish OKR의 핵심은 물고기를 잡고있다. 이 부분에서, 우리는 당신이 단계적으로 표시됩니다. 우리기구는 언급된 다른 유사한 다음과 같은 부분을 작곡. 원형 물고기 챔버 외부 직경 7.0 cm, 내경 6.5 cm 높은 7.0 cm입니다. 생선 실 주위에 드럼 밖으로 직경 8.5 cm, 내부 직경 7.5 cm 높은 7.0 cm입니다. 그것은 시계 방향으로 20의 속도로 시계 반대 방향으로 회전 수 분마다 ~ 70 회전 (RPM). 검은색과 흰색 줄무늬의 열 사이클은 드럼의 외부에 붙어 추적 종이에 그려져 있습니다. 조명 시스템은 21cm 직경의 두 원형 라이트 튜브로 구성되어 있습니다. <img src="/files/ftp_upload/1742/1742fig1.jpg" alt="그림 1" /> 그림 1. 스폰지의 모델.이 중간에 만들어진 절벽은 시야가 숨겨되지 않았는지 확인하는 것입니다. <ol><li> 로 Fig.1에 표시된 스펀지는 두 부분으로 구성되어 있습니다. 왼쪽 부분은 3cm X 폭 3cm X 높이 2cm 길이입니다. 오른쪽 부분은 길이가 3cm는 X 폭 3cm X 높이 3cm입니다. </li><li> 물고기 챔버의 바닥에 스펀지를 넣고 약 2 cm 깊이의 양육 물을 부어. 스폰지에 숨어 공기 방울은 사라졌어해야합니다. </li><li> 절벽 뒤에 2mm 정도 스폰지의 오른쪽 부분의 앞에 핀을 삽입합니다. </li><li> MS - 222의 성인 zebrafish를 마취. </li><li> 지느러미 최대로 스펀지에 zebrafish를 넣고 첫 번째 핀하기 전에 가슴 지느러미의 한쪽을 허용합니다. </li><li> 두 번째 핀은 다른 가슴 지느러미 뒤에 삽입됩니다. 그것은 첫 번째 핀 (그림도 참조하십시오. 2)와 협력하여 zebrafish 축소 시신을 피할 수 있습니다. </li><li> 동맥이 물고기 챔버에 준수는 고무의 조각 (모델링 점토)에 동맥 클램프 클램프 및 수정과 꼬리를 고정. </li><li> 세 번째와 네 번째 PIN은 물고기 신체의 cloacal 구멍 근처의 사이트에 심어져 있습니다. 그것은 심각하게 꼬리를 흔들 zebrafish을 방지 수 있습니다. </li><li> 다섯 번째 PIN은 여섯 번째 핀 그것의 반대편에있는 다음 첫 번째와 세 번째 핀 사이 sited입니다. 그들은 몸을 흔들어에서 zebrafish를 방지 수 있습니다. </li><li> 일곱째와 여덟째 PIN은 머리 방향을 유지하는 길 근처의 사이트에 삽입됩니다. </li><li> 물고기 챔버의 양육 물을 붓고. </li><li> zebrafish가 깨어나면 후, 5 ~ 10 분 정도 적응하자. </li><li> 이 장치의 빛이 스위치를 켜고 zebrafish 2 분 동안이 조명을 적응하자. </li><li> 격자를 운전하고 1 분 적응 zebrafish 수 있도록 모터에 스위치. </li><li> 카메라의 전원을 켜고 눈을의 움직임을 기록합니다. </li><li> 개인용 컴퓨터 (눈 각도 분석 대표 예제는 Fig.2에 표시됩니다)에 눈 각도 6 saccade 번호를 분석합니다. </li></ol><img src="/files/ftp_upload/1742/1742fig2.jpg" alt="그림 2" /> 그림 2. 안과 축 및 신체 축 사이의 각도. A)하면 격자의 방향은 비강 - 투 - 시간적이다, 우리는 부정적인 각도는 물고기 신체의 앞으로의 각도를 정의하고 생선 본문의 각도가 긍정적인 각도, 반대 (간략한 검토도 참조를 참조합니다. 6). 이 그림에서 숫자는 핀의 순서를 나타냅니다. 일반 그룹에서 한 생선 B) OKR 분석 예. 1 초 영화는 일련의 프레임에 전송되어 각 프레임의 눈 각도는 이미지 프로 플러스 소프트웨어로 측정되었다. 이 그림의 각 요점은 우리가 측정한 각 프레임을 나타냅니다. 참고 : <ol class="lalpha"><li> 모든 핀이 단지의 몸을 흔들어에서 zebrafish을 방지하는 데 사용됩니다, 너무 꽉 물고기에 해로운 것입니다. </li><li> 의 접촉 면적이 작은 같은 동맥 클램프가 꼬리를 손상 수 있습니다. 꼬리 무료되면,이 물고기를 수정하는 것은 매우 어렵습 S. 어떤 상태에서 권장 꼬리 주위에 붕대를 바꿈. </li><li> 아가미 근처의 사이트에 심어져 있습니다 일곱째와 여덟째 핀은 격자 그들의 시야에서 피난처해서는 안됩니다. </li></ol> 부 III. 애벌레 Zebrafish의 OKR 1. 메틸 셀룰로오스 오랫동안 살아남으 동안 애벌레 zebrafish는 6 % 메틸 셀룰로오스 용액에 고정하실 수 있습니다. 다음과 같이 메틸 셀룰로오스 용액을 준비합니다. <ol><li> 높은 수준에서 약 3 분 전자렌지에 넣어 익히지 200 ML ddH 2 O. </li><li> 감동 열판에 온천수를 전송하고 적극적으로 그것을 저어. </li><li> 점차 뜨거운 물에 12g의 메틸 셀룰로오스를 추가합니다. </li><li> 메틸 셀룰로오스가 잘 분산 후 3 분 저어 유지. </li><li> 그것이 분명해진다 때까지 얼음물과 솔루션을 쿨 다음 4를 장소 ° C. </li></ol><p클래스 = &quot;jove_step&quot;&gt; 2. 애벌레 Zebrafish <ol><li> 쌍이 남성과 수유 후 17:00 짝짓기 탱크에 성인 zebrafish의 여성. 다음날 아침은 신선한 짝짓기 탱크로 전송하고 1 시간 뒤에 배아를 수집합니다. </li><li> 14시 10분의 어두운주기 : 계란은 불빛과 28.5 ± 0.5 ° C가 인큐베이터에서 배아 매체에 보관됩니다. </li><li> 계란은 48 시간 포스트 수정 (HPF)에서 부화되며 OKR은 나중에 72 HPF하거나 테스트합니다. 이 비디오에서는 larvaes 5 DPF에 있습니다. </li></ol> 3. OKR 절차 <ol><li> CCD 카메라 및이 장치의 빛을 스위치를 갖춘 입체경 아래의 장치를 넣어. </li><li> 55 - mm 배양 접시에 20 ML 6 % 프로필 메틸 셀룰로오스를 추가합니다. </li><li> 이 접시의 중앙에 연속으로 여러 larvaes를 배치합니다. </li><li> 카메라 분야 1 μl의 볼륨에 대한 공기 방울을 넣어, 그것은 격자의 방향을 반영하는 거울로서 수 있습니다. </li><li> 모터에 스위치와 그들의 눈을의 움직임을 기록합니다. </li></ol> 부 IV. 성인 Zebrafish의 OMR 성인 zebrafish OMR 또한이 장치와 함께 진행 될 수 단지 (회전 속도는 흑백 사이클은 11아르 36 RPM이며, Fig.3a에서 보여준) 센터를 통해 물고기 수영을 방지하기위한 물고기 챔버에 칼럼을 넣으십시오. <ol><li> 이 생선 챔버에 물고기를 넣어. </li><li> 이 물고기 챔버의 중앙에 2cm 직경의 열을하라. </li><li> 둘 다 살아남 격자 및 순환 격자 한 분간 적응. </li><li> zebrafish의 움직임을 기록합니다. </li><li> 귀하의 개인 컴퓨터에 시간을 우연 속도를 분석 (예제 Fig.3b에 표시됩니다, 회전 속도가 36 RPM이며, 흑백주기 11) </li></ol><img src="/files/ftp_upload/1742/1742fig3.jpg" alt="그림 3" /> 그림 3. 성인 Zebrafish의 OMR. (A) 흰색 플라스틱 칼럼 그것에 걸쳐 물고기 수영을 방지하기위한 물고기 챔버의 중앙에 위치했다. (B) 성인 zebrafish의 우연 속도는 격자를 따라. 우연의 일치 비율은 총 녹음 시간은 다음 격자 시간의 비율로 정의했다. 50 % 가치는 100 % 잘 생선 다음과 같은 격자를 나타내며 물고기가 무작위로 수영장을 나타냅니다. 두 광학 신경은 10 초 정도 보석 포셉로 깔린했다. OMR의 동작은 호감 후 임의의 수영으로 나타났다.

<ol>
	<li>Beck, J., Gilland, E., Tank, D., Baker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+Ontogeny+of+Optokinetic+and+Vestibuloocular+Behaviors+in+Zebrafish+Medaka,+and+Goldfish.">Quantifying the Ontogeny of Optokinetic and Vestibuloocular Behaviors in Zebrafish Medaka, and Goldfish.</a> J Neurophysiol. 92, 3546-3561 (2004).</li><li>Brockerhoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+optokinetic+response+of+zebrafish+larvae.">Measuring the optokinetic response of zebrafish larvae.</a> Nature Protocols. 1, 2448-2451 (2006).</li><li>Emran, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OFF+ganglion+cells+cannot+drive+the+optokinetic+reflex+in+zebrafish.">OFF ganglion cells cannot drive the optokinetic reflex in zebrafish.</a> Proc Natl Acad Sci U S A. 104, 19126-19131 (2007).</li><li>Rinner, O., Rick, J., Neuhauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+Sensitivity,+Spatial+and+Temporal+Tuning+of+the+Larval+Zebrafish+Optokinetic+Response.">Contrast Sensitivity, Spatial and Temporal Tuning of the Larval Zebrafish Optokinetic Response.</a> Investigative Ophthalmology and Visual Science. 46, 137-142 (2005).</li><li>Roeser, T., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+Behaviors+in+Larval+Zebrafish+after+GFP-Guided+Laser+Ablation+of+the+Optic+Tectum.">Visuomotor Behaviors in Larval Zebrafish after GFP-Guided Laser Ablation of the Optic Tectum.</a> The Journal of Neuroscience. 23, 3726-3734 (2003).</li><li>Easter, S., Nicola, G. <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> Developmental Biology. 180, 646-663 (1996).</li><li>Maaswinkel, H., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+frequency+characteristics+of+the+optomotor+response+in+zebrafish.">Spatio-temporal frequency characteristics of the optomotor response in zebrafish.</a> Vision Research. 43, 21-30 (2003).</li></ol>

OKR의 정의에 따르면, 그것은 동물들이 저절로 회전 격자를 추구하기 위해 눈을 이동하는 현상입니다. 이 문제는 오랜 시간 (6 시간 이상 등)에 대해 실시되고있다면, 눈 근육이 피곤 수 있으며 OKR는 감쇠됩니다. 성인 zebrafish OMR은 다른 7 완벽하게 설명했습니다. 이 비디오에서는, 우리는 당신에게 우리의 장치와 함께 동작을 보여줍니다. 광학 신경이 짓눌린되면, 그들?...

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using the optokinetic response to study visual function of zebrafish

Optokinetic 응답 (OKR)은 동물이 주변에 회전 격자를 수행하기 위해 눈을 진동하는 동작입니다. 그것은 널리 애벌레 zebrafish 1-5의 시각 기능을 평가하기 위해 사용되고 있습니다. 그럼에도 불구하고, 애벌레 물고기를위한 표준 프로토콜은 아직 성인 zabrafish에 즉시 적용되지 않습니다. 여기, 우리 연구실에 설립하는 새로운 프로토콜을 사용하여 간단한 사용자 지정 - 내장된 장치와 성인 zebrafish의 OKR을 측정하는 방법을 소개합니다. 우리의 장치 및 성인 zebrafish에 OKR의 단계별 절차를 모두이 동영상에 그림입니다. 또한, 애벌레 OKR의 측정뿐만 아니라 optomotor 응답 (OMR) 성인 zebrafish의 시험은 또한이 동영상에서 시연하고 있습니다. 우리의 실험에서 성인 zebrafish의 OKR 분석은 최대 4 시간까지 마지막 수 있습니다. 성인 피시 비전 시스템을 조작할 때 성인 물고기에 적용 이러한 OKR 시험은보다 효율적으로 시각적 기능 조사에 도움이 될 것입니다. 스와 - 치 Zou과 우 인이 종이에 똑같이 기여.

Watch this Scientific Journal Video about Using the optokinetic response to study visual function of zebrafish at JoVE.com

Using the optokinetic response to study visual function of zebrafish

Optokinetic 응답 널리 애벌레 zebrafish의 시각 기능을 평가하기 위해 사용되고 있습니다. 그럼에도 불구하고, 애벌레 물고기를위한 표준 프로토콜은 아직 성인에 즉시 적용되지 않습니다<sup&gt; 1-5</sup&gt;. 여기, 우리 연구실에 설립하는 새로운 프로토콜을 사용하여 성인 zebrafish의 OKR을 측정하는 방법을 소개합니다.

zebrafish의 시각 기능을 연구하기 위해 optokinetic 응답을 사용하여

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

using-the-optokinetic-response-to-study-visual-function-of-zebrafish

Research

JoVE Journal

Biology

18.2K 조회수.  University of Science and Technology of China (USTC). Optokinetic 응답 널리 애벌레 zebrafish의 시각 기능을 평가하기 위해 사용되고 있습니다. 그럼에도 불구하고, 애벌레 물고기를위한 표준 프로토콜은 아직 성인에 즉시 적용되지 않습니다 1-5. 여기, 우리 연구실에 설립하는 새로운 프로토콜을 사용하여 성인 zebrafish의 OKR을 측정하는 방법을 소개합니다.

비디오: Using the optokinetic response to study visual function of zebrafish

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

유전자 기능을 분석 Zebrafish 배아들 중 Microinjection

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Zebrafish 상악 바벌의 연구 방법

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

안녕하세요 - C : Genomes의 3 차원 구조를 공부하는 방법.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Zebrafish의 Pronephros의 레이저 절제는 신장 상피 조직이 재생된를 공부

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish의 유충의 근육 기능을 평가하기 위해 수축하는 동안 힘 측정

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

높은 해상도 전체 마운트<em&gt; 제자리에서</em유전자 발현 및 기능을 연구하는 제브라 피쉬 배아에서&gt; 하이브리드

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

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 ...