우리는 사무엘 구청 감사, 다이앤 C.이 기술을 개척을위한 쉐익스과 그레고리 넬슨. 우리는 활성 마리의 정자 수야을 보여주는 동영상을위한 파반 Kadandale와 브라이언 Geldziler 감사, 도움이 토론에 대한 Singson 연구실의 구성원, CGC 종자와 NIH에 대한 권한을 부여 (R01HD054681)를 통해 우리를 지원하십시오.

남성 인구의 1) 농축 <ol><li> 실험 필요에 따라 남성의 큰 숫자는 다음 전략 중 하나를 채용하여 구할 수 있습니다 : <ol><li> 야생 타입 남성의 큰 인구는 NGM 판의 중앙에 OP50 씨앗의 작은 잔디 5 야생 타입 남성 1 남녀 추니를 건너하여 얻을 수 있습니다. 성공 세대의 약 50 %가 야생 타입 남성 것입니다. </li><li> 그를 - 5 (e1490) 또는 그를 - 8 (e1489) 나온것 그를 - 5. 남성의 큰 숫자를 던져 그를 - 8 남성 비옥하고 정자의 형태와 기능에 명백한 결함이 없습니다. 그래서, 그를 - 5 또는 그와는 - 8 남성은 많은 실험을위한 야생 타입 남성 대신에 사용할 수 있습니다. </li></ol></li></ol> 남성 2) 식별 및 격리 <ol><li> 벌레의 꼬리 형태를 검사하고, 남성 웜 꼬리가 반올림됩니다. </li><li> L4 무대 남성을 선택하고 E. 가진 NGM 접시 씨앗에게 전송 대장균은 그들이 하루나 이틀 성장 OP50 및하자. 나온것의 부재로 성장 독신 남성은 정자의 손실을 방지하고 따라서 spermatids의 많은 수의 실험 절차를 수행하는 동안 사용 가능한 것입니다. </li><li> 해부의 날, 세균없이 NGM 접시에 독신 남성을 전송하고 그들이 몇 분 동안 크롤 링을 보자. 이 단계는 E. 작은 레이어를 제거하는 데 도움이됩니다 대장균은 벌레의 표면에 준수. 또는 버퍼로 시계 유리 및 전송 남성의 M9 버퍼 (6g 나 2 HPO 4, KH 2 PO 3g 4 5g NaCl, 0.25g MgSO 리터 당 4 0.7 H 2 O)의 피펫 소량. </li></ol> 남성과 체외 활성화 3) 해부 <ol><li> 유리 슬라이드를 타고 자궁 펜을 사용하여 작은 원을 표시합니다. 피펫 1X 정자 매체 30-50 μl (50mM HEPES, 25mM KCl, 45mM NaCl, 1mM MgSO 4, 5mM CaCl 2, 10mM 덱스 트로 오스, 산도 7.8) 원 안에. 소수성 원은 그 경계 내에서 정자 매체를 유지하는 데 도움이됩니다. </li><li> 정자 매체에 50-10 수컷을 전송합니다. </li><li> 현미경 슬라이드를보기, 당신 손 모두에서 27 게이지 바늘로 연결된 두 주사기를 개최. </li><li> 벌레를 &#39;대기&#39;와 spermatids를 공개 남성의 뒷부분 끝을 잘라 다른 바늘을 사용하여 하나의 바늘을 사용합니다. </li><li> 벌레를 절단하는 것은 대개 즉시 spermatids의 수많은 퇴학. spermatids는 분 스테레오 현미경 &#39;과립&#39;로 볼 수 있습니다. 그러나, 때로는 일부 또는 spermatids의 대부분은 시체 내에 남아있을 수 있습니다. 이 경우에는 조심스럽게 유리 슬라이드 위에 시체 &#39;를 드래그하면&#39;시체에서 spermatids의 출시를 용이하게 수 있습니다. </li><li> spermatids를 활성화하려면, Pronase 전자 (20μg/ml)를 포함하는 1X 정자 중간에 벌레를 해부하다하고 5 분 동안 앉아 보자. </li><li> 슬라이드 실험의 시점에서 건조되는 것을 허용하지 않습니다. 필요한 경우 더 많은 1X 정자 매체를 추가합니다. </li></ol> spermtids와 마리의 정자 수야 4) 시각화 <ol><li> 부드럽게 해부 표본의 표면에 coverslip을 넣으십시오. </li><li> 낮은 배율 (10X)에서 spermatids 분야 찾기 </li><li> 보통 100X 기름 침지에서 - spermatids 또는 마리의 정자 수야의 세밀한 부분이 더 높은 배율로 이동하여 볼 수 있습니다. </li></ol> 5) 대표 결과 남성 C. 엘레간스으로부터 격리 spermatids의 DIC 이미지의 예제는 그림 1에 표시됩니다. Spermatids는 형태 구형이며 핵은 유수 있습니다. 체외 활성화의 정자에서는 그림 2에 표시됩니다. <img src="/files/ftp_upload/2336/2336fig1.jpg" alt="그림 1" /> 그림 1. C.의 DIC 이미지 엘레간스의 spermatids. <img src="/files/ftp_upload/2336/2336fig2.jpg" alt="그림 2" /> 그림 2. 체외 활성화 C로의 DIC 이미지 엘레간스은 마리의 정자 수야.

<ol>
	<li>Kimble, J., Crittenden, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+of+germline+stem+cells,+entry+into+meiosis,+and+the+Sperm/Oocyte+decision+in+Caenorhabditis+elegans.">Controls of germline stem cells, entry into meiosis, and the Sperm/Oocyte decision in Caenorhabditis elegans.</a> Annual Review of Cell and Developmental Biology. 23, 405-433 (2007).</li><li>Lhernault, S. W., Roberts, T. 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> Methods in Cell Biology. , 273-301 (1995).</li><li>Hodgkin, J., Horvitz, H., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondisjunction+Mutants+of+the+Nematode+Caenorhabditis+elegans.">Nondisjunction Mutants of the Nematode Caenorhabditis elegans.</a> Genetics. 91, 67-94 (1979).</li><li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+male+C.+elegans+anatomy.">Introduction to male C. elegans anatomy.</a> WormAtlas. , (2008).</li><li>Shakes, D., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+spermiogenesis+in+C.+elegans:+a+pharmacological+and+genetic+analysis.">Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis.</a> Dev Biol. 134, 189-200 (1989).</li><li>Nelson, G. A., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+fusion,+pseudopod+extension+and+amoeboid+motility+are+induced+in+nematode+spermatids+by+the+ionophore+monensin.">Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin.</a> Cell. 19, 457-464 (1980).</li><li>L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+cell+biology+of+spermatogenesis+in+the+nematode+C.+elegans.">The genetics and cell biology of spermatogenesis in the nematode C. elegans.</a> Molecular and Cellular Endocrinology. 306, 59-65 (2009).</li><li>Chatterjee, I., Richmond, A., Putiri, E., Shakes, D., Singson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+spe-38+gene+encodes+a+novel+four-pass+integral+membrane+protein+required+for+sperm+function+at+fertilization.">The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization.</a> Development. 132, 2795-2808 (2005).</li><li>Xu, X., Sternberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+elegans+sperm+TRP+protein+required+for+sperm-egg+interactions+during+fertilization.">A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization.</a> Cell. 114, 285-297 (2003).</li><li>Nelson, G., Roberts, T., Ward, S. <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+spermatozoan+locomotion:+amoeboid+movement+with+almost+no+actin.">Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin.</a> J Cell Biol. 92, 121-131 (1982).</li><li>Machaca, K., DeFelice, L. J., L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chloride+channel+localizes+to+Caenorhabditis+elegans+spermatids+and+chloride+channel+blockers+induce+spermatid+differentiation.">A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation.</a> Dev Biol. 176, 1-16 (1996).</li><li>LaMunyon, C., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+viability+of+mutant+and+manipulated+sperm+by+artificial+insemination+of+Caenorhabditis+elegans.">Assessing the viability of mutant and manipulated sperm by artificial insemination of Caenorhabditis elegans.</a> Genetics. 138, 689-692 (1994).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Spe 돌연변을 분석뿐만 아니라,이 프로토콜은 노화와 정자의 형태를 분석과 같은 다른 중요한 응용 프로그램을 가지고 있습니다. 이 프로토콜을 사용 Spermatids과 격리 마리의 정자 수야는 야생 타입과 돌연변이 정자 8, 9, 슬라이드 10, 생리적 측정 9, 11, 또는 인공 수정 12 정자의 운동성의 immunostaining 같은 다른 스트림 실험에서 사용할 수 있습니다.

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isolation and in vitro activation of caenorhabditis elegans sperm

남성과 나온것은 선충류 C. 엘레간스에있는 두 자연스럽게 발견 성적 양식입니다. amoeboid 정자는 남성과 나온것 모두에 의해 생성됩니다. 300 - - gametogenesis의 이전 단계에서 나온것의 배아 세포는 정자의 제한 번호로 차별과 spermatheca라는 작은 &#39;가방&#39;에 저장됩니다. 나중에, 지속 oocytes 1 생산 나온것. 반면, 남성들은 성인에 걸쳐 독점적으로 정자 생산하고 있습니다. 남성은 전형적인 성인 웜 2 전체 세포의&gt; 50 %를 차지하고 그 많은 정자를 생산하고 있습니다. 따라서 남성의 정자를 분리하는 것은 나온것의에서보다 쉽습니다. 남성의 극소수는 자연의 X 염색체 3 자연 이외의 분리로 인해 생성됩니다. 보다 편리하게 남성 또는 그에게 상승을주는 돌연변이의 소개 (수컷의 높은 발병률) 표현형로 나온것 크로싱하면 그 중 하나가 남성 인구 3 풍부 수있는을 통해 전략의 일부입니다. 남성은 쉽게 꼬리 형태 4을 관찰하여 나온것 구별하실 수 있습니다. 남성 꼬리가 짝짓기 구조 둥근 반면 남녀 추니 꼬리는 지적이다. 꼬리를 잘랐다 남성의 생식 요로 내에 저장된 spermatids의 수많은 출시. 해부가 27 게이지 바늘을 사용하여 스테레오 현미경으로 수행됩니다. spermatids가 실제로 다른 세포와 연결되지 않은 상태 때문에, 유압 압력 spermatids이 남성을 포함한 신체의 내부 내용을 퇴학. 말스 직접 &#39;정자 보통&#39;의 작은 방울을 해부하고 있습니다. Spermatids는 산도의 변경에 민감합니다. 따라서 HEPES, 좋은 버퍼링 용량을 가진 화합물은 정자 미디어에서 사용됩니다. 포도당과 정자 매체에있는 다른 소금은 정자의 무결성을 유지하기 위해 삼투압을 유지할 수 있도록 도와주십시오. 마리의 정자 수야에 spermatids의 포스트 meiotic 분화는 spermiogenesis 또는 정자의 정품 인증을 칭했다있다. 5 쉐익스하고, 넬슨 6 이전 라운드 spermatids 여기서 우리는 C.의 체외 spermiogenesis에서 보여주는 Pronase E. 등 다양한 활성화 화합물을 추가하여 마리의 정자 수야으로 차별화할 수있는 유도했다 Pronase E.을 사용 엘레간스의 spermatids 성공 spermiogenesis 임신을위한 필수 조건이며 따라서 spermiogenesis에 결함이 돌연변이 멸균됩니다. 지금까지 여러 가지 돌연변이가 특별히 spermiogenesis 과정 7 결함이 표시되었습니다. 이상은 소설 Spe (Spermatogenesis 결함)의 체외 활성화에 돌연변이 우리가이 행사에 참여 추가 선수를 발견할 도움이 될 중에 발견했습니다.

Watch this Scientific Journal Video about Isolation and In vitro Activation of Caenorhabditis elegans Sperm at JoVE.com

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

남성에서 spermatids을 분리하고 활성화하기위한 프로토콜<em&gt; C. 엘레간스</em&gt; 여기에 설명되어 있습니다. 남성 자료 spermatids의 뒷부분 끝을 절단. spermatids은 프로 테아제를 추가로 활성화할 수 있습니다.

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 ...

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Isolation and In vitro Activation of Caenorhabditis elegans Sperm

16.2K 조회수.  Rutgers University. 남성에서 spermatids을 분리하고 활성화하기위한 프로토콜 C. 엘레간스 여기에 설명되어 있습니다. 남성 자료 spermatids의 뒷부분 끝을 절단. spermatids은 프로 테아제를 추가로 활성화할 수 있습니다.

비디오: Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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 ...

연구

생물학

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 ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

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, ...

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, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

의 재배<em&gt; 예쁜 꼬마 선충</em&gt; 실험실에서 세 가지 차원에서

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

결합 된 뉴클레오티드와 꼬마 선 충 의 단백질 추출

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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 ...

분리 및 체외에서 활성화 Caenorhabditis 엘레간스 정자

분리 및 체외에서 활성화 Caenorhabditis 엘레간스* 정자*