우리는 헌터 연구소의 협력 자연을 인정하고,이 기술의 개발이 가능하고, 즐거운 한 도움이 토론과 도움에 감사합니다. 나이젤 Delaney와 웜과 박테리아 변종에 대한 Caenorhabditis 유전학 센터를 감사드립니다. 이 작품은 건강의 국립 연구소 CPH에 GM089795 보조금에 의해 지원되었다.

<ol>
	<li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Stable+Transgenic+C.+elegans+Using+Microinjection.">Generation of Stable Transgenic C. elegans Using Microinjection.</a> J. Vis. Exp. (18), e833-e833 (2008).</li><li>Hirose, T., Nakano, Y., Nagamatsu, Y., Misumi, T., Ohta, H., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+GMP-dependent+protein+kinase+EGL-4+controls+body+size+and+lifespan+in+C+elegans.">Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans.</a> Development. 130, 1089-1099 (2003).</li><li>Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+cell+lineage+following+laser+ablation+of+cells+in+the+somatic+gonad+of+Caenorhabditis+elegans.">Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans.</a> Dev. Biol. 87, 286-300 (1981).</li><li>Hegreness, M., Shoresh, N., Hartl, D., Kishony, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+equivalence+principle+for+the+incorporation+of+favorable+mutations+in+asexual+populations.">An equivalence principle for the incorporation of favorable mutations in asexual populations.</a> Science. 311, 1615-1617 (2006).</li><li>Labrousse, A., Chauvet, S., Couillault, C., Kurz, C. L., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+is+a+model+host+for+Salmonella+typhimurium.">Caenorhabditis elegans is a model host for Salmonella typhimurium.</a> Curr. Biol. 10, 1543-1545 (2000).</li><li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome. Biol. 2, (2001).</li><li>Min, K., Kang, J., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+feeding+RNAi+method+for+simultaneous+knock-down+of+more+than+one+gene+in+Caenorhabditis+elegans.">A modified feeding RNAi method for simultaneous knock-down of more than one gene in Caenorhabditis elegans.</a> Biotechniques. 48, 229-232 (2010).</li><li>Zhuang, J. J., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specificity+of+Caenorhabditis+elegans+Enhanced+RNAi+Mutants.">Tissue-specificity of Caenorhabditis elegans Enhanced RNAi Mutants.</a> Genetics. 188 (1), 235-237 (2011).</li><li>Way, J. C., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mec-3,+a+homeobox-containing+gene+that+specifies+differentiation+of+the+touch+receptor+neurons+in+C.+elegans.">mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans.</a> Cell. 54, 5-16 (1988).</li><li>Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Bot, N. L. e., Moreno, S., Sohrmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421, 231-237 (2003).</li><li>Winston, W. M., Molodowitch, C., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+RNAi+in+C.+elegans+requires+the+putative+transmembrane+protein+SID-1.">Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1.</a> Science. 295, 2456-2459 (2002).</li></ol>

관심 없음 충돌이 선언 없습니다.

여기 모델 유기체 C.에서 세포 외액의 절연 및 특성을 수있는 새로운 방법을 발표 elegans. 기술은 세포 외액 자신의 총 볼륨을 높이기 위해 기증 웜의 유전자 또는 물리적 조작을 시작합니다. 세포 외액은 다음 수정 microinjection 기술을 사용하여 절연하고 있습니다. 웜은 절차를 수행하는 동안 여전히 벌레를 잡아 건조 아가로 오스 패드를 사용하여 microinjection에 장착되어 있습니다. 패?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; 일</strong</td&gt;<td<strong&gt; 웜 준비</strong</td&gt;<td<strong&gt; 재료 준비</strong</td&gt;</tr&gt;<tr&gt;<td&gt; -7 사전</td&gt;<td&gt; 깨끗한 유지 잘 공급<em&gt; CLR-1 (e1745)</em&gt;와 N2 웜</td&gt;<td&gt; 사출 패드, NGM 플레이트, NGM + Carb / IPTG 접시, OP50 LB 주식을 만들기</td&gt;</tr&gt;<tr&gt;<td&gt; -6</td&gt;<td</td&gt;<tdLB에 대한 RNAi 음식 아웃&gt; 승리 + Carb</td&gt;</tr&gt;<tr&gt;<td&gt; -5</td&gt;<td</td&gt;<td&gt; RNAi 음식 박 3 ML의 예방</td&gt;</tr&gt;<tr&gt;<td&gt; -4</td&gt;<td&gt; RNAi 판에 배아 이동</td&gt;<td&gt; 종자 RNAi 판</td&gt;</tr&gt;<tr&gt;<td&gt; -3</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; -2</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; -1</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 0</td&gt;<td&gt; 4 시간 동안 25 웜를 Shift</td&gt;<td&gt; 검정 접시 만들기</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; NGM 플레이트를 청소 벌레를 이동</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; Vampiric 전송</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; 수신자를 복구</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 1</td&gt;<td&gt; 이전 수신자를 제거</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 2</td&gt;<td&gt; 점수 자손</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt; 표 1.</strong&gt; 재료 준비 일정.</p&gt;<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; 장비</strong</td&gt;<td<strong&gt; 회사 소개</strong</td&gt;<td<strong&gt; 카탈로그 번호</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Picoliter 압력 주입기</td&gt;<td&gt; 워너 계측기</td&gt;<td&gt; 65-0001 (PLI-100)</td&gt;</tr&gt;<tr&gt;<td&gt; 불타는 / 브라운 micropipette 풀러</td&gt;<td&gt; 셔터 계측기</td&gt;<td&gt; P97</td&gt;</tr&gt;<tr&gt;<td&gt; Axiovert 200</td&gt;<td&gt; Zeiss</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td colspan="3"<strong&gt; 시약</strong&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; 미네랄 오일</td&gt;<td&gt; EM 과학</td&gt;<td&gt; MX1560-1</td&gt;</tr&gt;<tr&gt;<td&gt; 22x50없이 1 ½ 커버 유리</td&gt;<td&gt; 코닝</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; SeaKem LE 아가로 오스</td&gt;<td&gt; Lonza</td&gt;<td&gt; 50,004</td&gt;</tr&gt;<tr&gt;<td&gt; 붕규산 유리 모세 혈관</td&gt;<td&gt; 세계 Precisions 계측기</td&gt;<td&gt; 1B100F-4</td&gt;</tr&gt;<tr&gt;<td&gt; 차아 염소산 나트륨 솔루션 (5 % 유효 염소)</td&gt;<td&gt; JT 베이커</td&gt;<td&gt; 9416-01</td&gt;</tr&gt;<tr&gt;<td colspan="3"<strong<em&gt; C. elegans</em&gt;와 박테리아 변종</strong&gt;</td&gt;</tr&gt;<tr&gt;<td<em&gt; CLR-1 (e1745) II</em&gt;<sup&gt; 9</sup</td&gt;<td&gt; Caenorhabditis 유전학 센터 (CGC)</td&gt;<td&gt; CB3241</td&gt;</tr&gt;<tr&gt;<td<em&gt; glp-1</em&gt; RNAi 벡터<sup&gt; 10</sup&gt;</td&gt;<tdBioscience&gt; 소스 (Ahringer 수유 도서관)</td&gt;<td&gt; F02A9.6</td&gt;</tr&gt;<tr&gt;<td<em&gt; PAL-1</em&gt; RNAi 벡터<sup&gt; 11</sup&gt;</td&gt;<td&gt;</td&gt;<td&gt; pHC187</td&gt;</tr&gt;<tr&gt;<td&gt; OP50-GFP<sup&gt; 5</sup&gt;</td&gt;<td&gt; Caenorhabditis 유전학 센터 (CGC)</td&gt;<td&gt; OP50-GFP</td&gt;</tr&gt;<tr&gt;<td&gt; YFP<em&gt; E. 대장균</em&gt;<sup&gt; 4</sup&gt;</td&gt;<td&gt;</td&gt;<td&gt; MC4100-YFP</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt; 표 2.</strong&gt; 특정 시약 및 장비.</p

vampiric isolation of extracellular fluid from caenorhabditis elegans

유전자 다루기 쉬운 모델 생물 C. elegans의 성장과 작은 크기의 용이성, 그 짧은 세대 시간으로 활성화, 생물의 질문 무수히으로 통찰력을 제공하고 있습니다. 이 작은 크기는하지만 다른 모델 시스템에서 발견 된 기술적 접근 방법의 수를 금지하고 있습니다. 예를 들어, 포유류의 시스템과 식물의 접목 기술의 수혈은 순환 시스템 조성과 신호의 질문을 수 있습니다. 최근 직접 분석에 불가능 때까지 웜의 순환 시스템 pseudocoelom이 있습니다. 세포 신호 및 순환 시스템을 구성 C.의 질문에 답변하려면 elegans 연구원은 전통적으로 유전자 분석, 세포 / 조직 특정 구조, 그리고 모자이크 분석 해 나가고 있습니다. 이 기술은 세포 사이에 어떤 일이 일어나고 있는지를 추론 할 수있는 수단을 제공하지만, 세포 분자의 식별 및 특성에 보편적으로 적용되지 않습니다. 여기 NE을 제시직접 검정 C.의 pseudocoelomic 유체에 wly 개발 기술 elegans. 기술은 세포 외액의 볼륨을 높이기 위해 중 유전 적 또는 물리적 조작으로 시작됩니다. 이후 동물은 고급 밸런스 압력 제어 할 수있는 microinjection의 장비를 사용하여 vampiric 역방향 microinjection 기술을 받게됩니다. 세포 외액의 분리 후, 수집 된 유체가 다른 동물로 전송 또는 분자에 의해 assayed 할 수 있습니다. 이 기법의 효과를 입증하기 위해 우리는 체계적 RNAi 반응 동안 분석 세포 신호 분자, 긴 dsRNA의 특정 예를에 자세한 방법을 제시한다. 체계적 RNAi의 특성은 원리 예제의 증거이지만, 우리는 순환 시스템 조성과 신호의 질문에 다양한 대답을 적응 것으로이 기법을 참조하십시오.

Watch this Scientific Journal Video about Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans at JoVE.com

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

모델 생물<em&gt; C. elegans</em&gt; 수동 순환 시스템으로 pseudocoelomic 유체를 사용합니다. 이 유체의 직접 분석은 이전에 수 없었습니다. 여기에 직접 검정 세포 공간을에 소설 기법을 제시하고, 원칙 예제의 증거로 RNAi 반응하는 동안 조직 입을 신호를 사용합니다.

에서 세포 외액의 Vampiric 절연<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

vampiric-isolation-of-extracellular-fluid-from-caenorhabditis-elegans

Research

JoVE Journal

Biology

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

10.1K 조회수.  Harvard University. 모델 생물 C. elegans 수동 순환 시스템으로 pseudocoelomic 유체를 사용합니다. 이 유체의 직접 분석은 이전에 수 없었습니다. 여기에 직접 검정 세포 공간을에 소설 기법을 제시하고, 원칙 예제의 증거로 RNAi 반응하는 동안 조직 입을 신호를 사용합니다.

비디오: Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

측정 Caenorhabditis 엘레간스</em솔리드 미디어> 수명

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

연구

생물학

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

분리 및 체외에서</em의> 활성화 Caenorhabditis 엘레간스 정자

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

솔리드 플레이트 기반식이 제한에 Caenorhabditis 엘레간스</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

에서 프로스타글란딘 추출 및 분석<em&gt; Caenorhabditis의 엘레</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

에있는 고도 불포화 지방산의식이 보충제<em&gt; 예쁜 꼬마 선충</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

그대로 꼬마 선 충 에 산소 소비 속도의 측정

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

예쁜꼬마선충의 정화 및 분석 세포외 소포

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Caenorhabditis 예인단에서 조직 특정 전립선 성 감소를 정량화

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

에서 세포 외액의 Vampiric 절연

요약

더 많은 비디오 탐색

이 비디오의 챕터

측정 Caenorhabditis 엘레간스 수명

분리 및 체외에서 활성화 Caenorhabditis 엘레간스* 정자*

솔리드 플레이트 기반식이 제한에 Caenorhabditis 엘레간스

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

기본

에서 프로스타글란딘 추출 및 분석

에있는 고도 불포화 지방산의식이 보충제

그대로 꼬마 선 충 에 산소 소비 속도의 측정

예쁜꼬마선충의 정화 및 분석 세포외 소포

Caenorhabditis 예인단에서 조직 특정 전립선 성 감소를 정량화

에서 세포 외액의 Vampiric 절연

요약

더 많은 비디오 탐색

이 비디오의 챕터

측정 Caenorhabditis 엘레간스 수명

분리 및 체외에서 활성화 Caenorhabditis 엘레간스 정자

솔리드 플레이트 기반식이 제한에 Caenorhabditis 엘레간스

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

기본

에서 프로스타글란딘 추출 및 분석

에있는 고도 불포화 지방산의식이 보충제

그대로 꼬마 선 충 에 산소 소비 속도의 측정

예쁜꼬마선충의 정화 및 분석 세포외 소포

Caenorhabditis 예인단에서 조직 특정 전립선 성 감소를 정량화

분리 및 체외에서 활성화 Caenorhabditis 엘레간스* 정자*