우리는 그의 가치가 도와 주셔서 감사합니다 드르 안토니오 미란다 - Vizuete 싶습니다. 우리는 또한 가치있는 기술 지원을 살마 Boulayoune와 이사벨 카발 레로 우리의 감사를 표현하는 것을 좋아합니다. 이 작품은 준타 데 안다 루시아 (BIO - 272)에서 부여로 재정되었다. 이 연구는 유럽에서 유전자 실험을 규제하는 현행 법률에 계약에서 실시되었습니다.

1 : 삼투 회피 분석. <ol><li> 약 16~24시간 분석하기 전에, OP50 E.를 포함하는 새로운 NGM 접시 각 유전자형의 L4 애벌레 무대 동물을 선택 대장균은 20 ° C.에 그것을 andincubate 다음 날 젊은 성인과 함께 실험을 시작합니다. </li><li> 이것은 분석 &quot;맹목적&quot;을 수행하는 것이 좋습니다. assayed하는 각각의 응력과 접시는 실험이 완료되면 가면 될 relabelled 접시를 사용하여 분석을 수행하는 두 번째 실험자에 의해 relabelled해야합니다. </li><li> 분석의 일, 1 % 콩고 레드 솔루션 Fructose와의 4M 재고 솔루션을 준비하고 실온에서 완전히 디졸브. 염색 시간과 침전 수 있으므로 우리는 각 분석하기 전에 해결책을 확인하는 것이 좋습니다. </li><li> NGM 플레이트에 대한 환상 링 (1cm 직경)은 붉은 4M의 Fructose와 솔루션을 15 μl와 고체 배지의 중앙에 설명되어 있습니다. Fructose와 솔루션 한천에 흠뻑 젖어 보자, 이것은 보통 2 5 분 사이에 걸립니다. </li><li> 반지 내의 각 변형의 개별 젊은 성인 동물을 플레이스와 삼투 장벽에 대한 응답을 결정하기 위해 다음 10 분 이상하십시오. 연속으로 여섯 번 이상의 반지를 피하 동물은 정상으로 분류되며 미만보다 여섯 시도에 반지를 종료들은 삼투 감도에 결함이있는 것으로 간주됩니다. 참고 : 제어 변형 각 분석에 사용해야합니다. 그들이 결정적으로 반지의 장벽을 피하기로 N2 젊은 성인 동물 긍정적인 컨트롤로 사용됩니다. 이전 굶주린 Dauer 애벌레 통과하거나 너무 건조 접시에서오고있다 동물을 사용해서는 안됩니다. 처음에는, 컨트롤 동물은 분석 접시와 솔루션이 올바른지 확인하기 위해 중복으로 평가해야합니다. </li></ol> 2 : RNAi 먹이로 최저의 벌레의 생성. <ol><li> RNAi 번호판 : NGM RNAi의 먹이 접시는 리터 당 포함 17g의 한천은 2,5 g 펩톤, 3.0 g NaCl, 5 MG ML -1 콜레스테롤 1 ML는, H 2 O 및 압력솥과 1리터로 술병을 입력합니다. 한천은 약 65 ° C 냉각 후, 1 M KPO 4, 산도 6.0, 1 M CaCl 2, 1M MgSO 4, 0,5 ML carbenicillin (50 MG ML -1)과 1 개 중 1 개의 ML 1 ML 25 ML을 추가 ML 1M의 IPTG. 당신이 준비한 탄탄한 NGM 시작하는 경우, microwaving에 의해 고체 매체를 녹으 후 시원한로 누른 다음 다른 이름으로 이전에 표시 KPO 4, 산도 6.0, CaCl 2, MgSO 4, carbenicillin과 IPTG을 추가하기 위해 벤치에 액체 NGM를 놓으십시오. 각 60mm 페트리 접시에 NGM 10 ML을 추가합니다. 냉각을 허용하고 사용하기 전에 밤새 RT에서 그들을 유지하는 번호판을 반전. 플레이트는 약 4-5 일 동안 4 ° C에서 비닐 봉지에 저장할 수 있습니다. </li><li> 박테리아 준비와 유도 : Luria - Bertani (LB) 한천 플레이트 (17g의 한천, 10g tryptone, 5g에, 대상 유전자에 해당하는 조각을 포함하는 L4440 벡터로 변형 E.coli HT115 (DE3) 변형의 식민지를 분리 효모 추출물, 그리고 리터 당 10g NaCl) 암피실린 (50 μg / ML)과 tetracyclin (15 μg / ML)를 포함. 박테리아의 식민지 선택 50 μg / ML 암피실린와 LB에 접종하고, 37 잡고 6 8 H에 대한 성장 ° C; 준비 NGM 판에 씨앗이 문화의 한 방울과 야간 잠복기되기 전에 철저하게 접시를 건조 ( 상온 12 24 H)은 박테리아가 성장하도록하고 유도를 시작하십시오. 테트라 사이클린은 민감한 빛이며 접시 사이에 RNAi의 assays의 다양성에 영향을 미칠 수 있기 때문에 부화가 어둠 속에서되었습니다. 그것은 긍정적이고 RNAi 먹이 실험에 대한 부정적인 컨트롤을 사용하는 것이 필요합니다. 긍정적인 컨트롤은 UNC - 22 유전자 시퀀스를 포함하는 벡터로 변환 L4440 E.coli 세포 HT115입니다. UNC - 22 유전자의 분해는 &quot;꿈틀 표현형&quot;를 생산하고 있습니다. 대조군은 빈 L4440 벡터로 변형 E.coli 세포 HT115입니다. </li><li> 웜 처리 및 채점 : 20 박테리아와 부화하지 않고 NGM 접시에 OP50와 씨앗 NGM 플레이트에서 첫 날, 장소 L4 무대 웜 ° 12 시간 C (금식). 다음날, 특정 타겟 유전자 RNAi을 표현 박테리아와 seedded 접시에 젊은 금식 성인 나온것을 전송할 수 있습니다. 20 40~48시간를 남겨 ° C는 F1 자손을 생성합니다. 그런 다음 같은 박테리아와 씨앗을 다른 접시에 몇 F1 어른 벌레를 놓습니다. 24-48시간 후, 선택하고 F2 자손, 젊은 성인 (몇 계란과 완벽하게 형성 튀어나온 산문)과 phenotypes에 대한 점수에서 분리. 참고 :. 뉴런의 RNAi 유도 때문에 RNAi에 C 엘레간스 신경 계통의 내화물 속성 중 일부 제한이 있습니다. 신경이 비효율의 문제를 극복하기 위해 그것은 rrf - 3 변형, 뉴런 10 RNAi의 효과에 과민 배경을 사용하는 것이 좋습니다. 실험없이 differenc에서에스이 유전자에 대한 브리스톨 N2 및 rrf - 3 변종 사이의 발견과 표현형 분석을했다. </li></ol> 3 계통 사용됩니다. C. 이 작품에 사용된 엘레간스의 종자는 표 1에 표시됩니다. 돌연변이 변종은 밖으로 넘어 N2에 대한 야생 했어요 - 입력 돌연변이 유발 프로토콜 중에 생성된 것일 수도 원치 않는 임의의 변이를 제거하려면 적어도 네 번. .. OP50 E 대장균의 변종은 플라스미드 nrx - 1을 들고 pL4440 (JA : C29A12.5)로 Caenorhabditis 유전자 센터, 미네소타 대학, 미국 E.coli HT115 (DE3)에 의해 suppliied되었으며 nlg - 1 (JA : C40C9.5 ) 유전자 조각은 박사 피터 Askjaer, 센트 Andaluz 호텔 드 Biología 델 Desarrollo (CABD), CSIC 대학교 파블로 Olavide, 세비야, 스페인에 의해 제공되었다. 4 : 대표 결과. 설명의 결과는 그림 1과 2에 표시됩니다. 열 각 변형의 동물과 세 복제 실험 최소가 진행되었다. <img alt="그림 1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> 그림 1. 삼투 회피 동작의 실험. 제어 변종과 nrx - 1 V 결함 mutantworms들이 삼투 장벽 (4M Fructose와)을 발생하는 경우 거꾸로 반대로 대응 (ok1649 및 tm1961). nlg - 1 (ok259 및 tm474) X 돌연변이 장벽을 감지하기 위해 실패합니다. VX 및 CRR23 (tm1961, ok259); nlg - 1 nrx - 1, 종자 CRR21 (ok259 ok1649)에 결함 더블 돌연변 VX는 야생 타입 표현형를 발견 했어요. *는 t - 학생 시험에 의해 브리스톨 N2 야생 형 각 변형의 반응에 T - 학생 검사로 큰 차이 (P ≤ 0.001)를 나타냅니다 <img alt="그림 2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> 그림 2. RNAi 먹이로 최저의 벌레와 실험. E.coli HT115 (DE3)는 빈 pL4440 벡터로 변환하거나 nrx이 - 1 또는 nlg - 1 먹이를 다른 웜 변종에 사용했던 대상 유전자에 해당하는 조각을 포함하는. *이 비어 pL4440 벡터와 공급 N2 스트레인과 비교 nlg - 1 유전자를 대상으로 조각으로 pL4440 벡터를 옮기는 박테리아와 공급 N2 변형의 반응에 T - 학생 검사로 큰 차이 (P ≤ 0.001)를 나타냅니다 .

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Neurexins 및 neuroligins는 시냅스 전송 11 시냅스 연결 12 분화에 필수적인 역할을 수행합니다. 두 분자가 자폐증 13,14에 대한 후보 유전자로 확인되었습니다. 이 비디오에서 우리는 우리가 C로 삼투 회피 반응에 영향을 미치는 유전자의 효과를 연구 수있는 간단한 방법을 보여 엘레간스. Neuroligin 결함 돌연변이 삼투 강도를 감지에 결함이되며...

표 1. C. 엘레간스의 변종. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 변형 </td><td> 유전자 </td><td> Allele </td><td> 출처 </td></tr><tr><td> 브리스톨 N2 </td><td> - </td><td> - </td><td> aCGC </td></tr><tr><td> VC228 </td><td> nlg - 1 </td><td> ok259 </td><td> CGC </td></tr><tr><td> FX00474 </td><td> nlg - 1 </td><td> tm474 </td><td> bNBP - 일본 </td></tr><tr><td> VC1416 </td><td> nrx - 1 </td><td> ok1649 </td><td> CGC </td></tr><tr><td> FX1961 </td><td> nrx - 1 </td><td> tm1961 </td><td> NBP - 일본 </td></tr><tr><td> NL2099 </td><td> rrf - 3 </td><td> pk1436 </td><td> CGC </td></tr><tr><td> CRR21 </td><td> nrx - 1, nlg - 1 </td><td> ok1649; ok259 </td><td> 이 작품은 </td></tr><tr><td> CRR22 </td><td> nrx - 1, nlg - 1 </td><td> tm1961, ok259 </td><td> 이 작품은 </td></tr></tbody></table> A. Caenorhabditis 유전자 센터, 미네소타 대학, 미국. B. 실험 동물 &quot;선충류 C. 엘레간스&quot;에 대한 국립 Bioresource 프로젝트. 도쿄 여자 의과 대학, 일본.

osmotic avoidance in caenorhabditis elegans: synaptic function of two genes, orthologues of human nrxn1 and nlgn1, as candidates for autism

Neurexins 및 neuroligins은 세포 접착 분자는 흥분성의 억제와 시냅스에 존재하고, 그들이 올바른 신경망 기능 1 필수입니다. 이러한 단백질은 presynaptic 및 postsynaptic 세포막 2 볼 수 있습니다. 생쥐의 연구 neurexins과 neurologins가 시냅스 전달 1 필수 역할을 나타냅니다. 최근 보고서는 인간의 신경 시스템의 개발 기간 동안 변경의 연결을 연결 자폐증 스펙트럼 장애 3 가지 경우 많은 병인의 기초를 구성 수있는 것으로 나타났습니다. Caenorhabditis의 엘레간스 때문에 실험실 실험에 대한 단순, 시냅스 구성 요소의 기능 연구를 촉진하기위한 실험 도구로 사용하며, 신경 시스템과 신경 배선이 완전히 특징되었음을 주어진 수 있습니다. C에서. 엘레간스 nrx - 1 nlg - 1 유전자는 각각 알파 - neurexin - 1 neuroligin - 1 단백질, 인코딩 인간 NRXN1 및 NLGN1 유전자에 orthologous 있습니다. 인간과 nematodes에서 neurexins 및 neuroligins의 조직 기능 도메인에 대한 존중과 유사합니다. 선충류의 머리 amphid, 삼투 강도를 포함한 다양한 자극에 반응을 mediates 선충류의 감각 기관이 포함되어 있습니다. amphid은 솜털이있는 dendrites 한 presynaptic 터미널 축삭 4 12 감각 뉴런의 바이폴라 이루어집니다. ASHR 및 ASHL라는 이러한 뉴런의 두 높은 삼투 강도 5 수용성 repellents를 감지, 삼투 감각 기능에 특히 중요합니다. 이 두 뉴런의 dendrites는 입 끝에으로 길이 그들이 행동 반응 6 결정 다른 뉴런과 시냅스 연결을 어디에 axons은 신경 링을 확장할 수 있습니다. 높은 삼투 강도 회피에 neurexin 및 neuroligin의 의미를 평가하기 위해, 우리는 4M Fructose와 반지 7 기반 방법을 사용, nrx - 1 nlg - 1 유전자에 결함이 C. 엘레간스 돌연변이의 다른 응답을 보여줍니다. 행동 phenotypes는 특정 RNAi의 클론 8을 사용하여 확인되었다. C로 엘레간스는 RNAi를 실행하는 데 필요한 dsRNA는 9 먹이에서 관리하실 수 있습니다. 음식을 통해 dsRNA의 납품 따라서 유전자 구성 요소와 네트워크 경로의 신분을 허용 관심있는 유전자의 RNAi 간섭을 유도.

Watch this Scientific Journal Video about Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism at JoVE.com

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins 및 neuroligins는 시냅스 차별화 및 전송에 필수적인 역할을 수행하는 막 - 신경 세포의 접착 단백질입니다. 의 Neuroligin 부족한 돌연변이<em&gt; C. 엘레간스</em&gt;는 삼투 강도를 감지에 결함이 있지만, 그들은 또한 neurexin 코딩 유전자에 돌연변이 포함된 경우, 그들은 야생 유형 표현형을 복구합니다.

의 삼투 회피 Caenorhabditis 엘레간스 : 두 유전자의 시냅틱 기능, 인간의 Orthologues NRXN1과 NLGN1</em자폐증을위한>로 후보

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

osmotic-avoidance-caenorhabditis-elegans-synaptic-function-two-genes

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JoVE Journal

Biology

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

11.7K 조회수.  Facultad de Ciencias, Universidad de Córdoba. Neurexins 및 neuroligins는 시냅스 차별화 및 전송에 필수적인 역할을 수행하는 막 - 신경 세포의 접착 단백질입니다. 의 Neuroligin 부족한 돌연변이 C. 엘레간스는 삼투 강도를 감지에 결함이 있지만, 그들은 또한 neurexin 코딩 유전자에 돌연변이 포함된 경우, 그들은 야생 유형 표현형을 복구합니다.

비디오: Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

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 ...

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 ...

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 ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

에서 지방 질량의 생화학 및 높은 처리량 미세 평가<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

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, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

샘플 준비 및 전송 전자 현미경 검사 법에 의해 Exosomes의 영상

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

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 ...

의 삼투 회피 Caenorhabditis 엘레간스 : 두 유전자의 시냅틱 기능, 인간의 Orthologues NRXN1과 NLGN1로 후보