저자 분사 마커에 대한 dbl-1 cDNA와 닥터 크리스토퍼 Rongo (Waksman 연구소, Rutgers 대학, 뉴저지)의 선물 박사 릭 Padgett (Waksman 연구소, Rutgers 대학, 뉴저지)에 감사드립니다. 닥터 바스 그랜트의 연구실에서 GFP-태그를 dbl-1 구조의 낮은 사본 번호 통합을위한 유전자 총기 포격을 수행. 르네 가르시아 연구실 texIs100의 창출 동안 기술 지원을 제공했습니다. 르네 가르시아, 로빈의 Lints 및 Hongmin 진 실험실 생산적인 조언을 제공했습니다. 이 작품은 분자 및 세포 의학 TAMHSC학과 시동 기금에 의해 자금 지원되었다. 화합물 범위와 회전 디스크 공촛점는 부서와 딘의 의학 사무소의 TAMHSC 대학에서 제공하는 자금으로 구입했습니다.

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우리는 공개 할게 없다.

여기서 제시 RNAi 심사 방법은 정상적인 (또는 형질) postembryonic 표현형에 필요한 유전자 제품의 민감하고 빠른 분석을 가능하게합니다. 표시 예제 fluorescently 태그가 단백질의 subcellular 지방화에 관련된 유전자에 대한 화면입니다. 그러나,이 프로토콜은 관심사의 다른 postembryonic phenotypes에 영향을 미치는 유전자를 식별하기 위해 수정할 수 있습니다. 이 방법은 RNAi 라이브러리를 사용하여 후보 유전자 접근 방법을 이용합니다. 돌연변이 유발 기술을 사용하여 앞으로 유전 화면 무작위로 유도된 소설 대립 유전자를 식별하지만, 이러한 대립 유전자는 24을 식별하는 데 상당한 시간과 노력, 그리고 / 또는 자금을 필요로합니다. cDNA 라이브러리로, 반면에 관심이 RNAi의 표현형 빠르게 후보 유전자를 확인 의한 cDNA 삽입 순서. 또한 복제 가능한 cDNA 삽입이 확인된 후보자의 다운 스트림 분석을 용이하게합니다. 변이된 때 치명적이다 또한, 유전자식별 및 RNAi를 사용하여 분석할 수 있습니다. 유도 박테리아에 부화 동물을 배치하면이 방법은 또한 중요한 배아 역할을 유전자에 대한 postembryonic 결함의 관찰을 허용합니다. 관찰된 동물 굶주려 있지 않은지 확인하기 위해주의를 기울입니다. 기아 동물의 표현형 영향을 미칠 수 있습니다. 소설 RNAi의 phenotypes는 굶주린 동물에서 확인된 경우, 표현형는 RNAi 아니라 기아의 결과임을 확인하기 위해 반복한다. 기아를 방지하기 위해 고정 단계 (성장 매체는 매우 &quot;구름&quot;입니다)에 재배되어 신선한 박테리아를 사용하고, 잘마다 ~ 30 동물을 사용합니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> NGM 한천 </td><td> 선충류 성장 매체 </td><td> IPM 과학, INC </td><td> NGM 한천 프로토콜 25 다음 준비할 수 </td></tr><tr><td> M9 중간 </td><td> 22mM KH 2 PO 4, 42mM 나 2 HPO 4, 86mM NaCl, 1 ㎜ MgSO 4 </td><td></td><td> 26 </td></tr><tr><td> 한천 - 한천 </td><td> EMD 화학 주식 회사 </td><td> 1.01614.1000 </td><td> NGM 플레이트 물에 2 %. 현미경 슬라이드 패드를위한 물 4 % (초기 및 이후 녹기 전자 레인지 압력솥). </td></tr><tr><td> Bacto 펩톤 </td><td> Becton 디킨슨 - Difco CP </td><td> 211,677 </td><td> 0.25 % </TD&gt; </tr><tr><td> IPTG </td><td> 연구 제품 국제 주식 회사 </td><td> I56000-5.0 </td><td> 1 ㎜ 최종 농도 </td></tr><tr><td> carbenicillin </td><td> 연구 제품 국제 주식 회사 </td><td> C46000-5.0 </td><td> 50 μg / ML 작업 희석 </td></tr><tr><td> LB 국물 레녹스 </td><td> Becton 디킨슨 - Difco CP </td><td> 240,230 </td><td> 20g / 리터 </td></tr><tr><td> 테트라 사이클린 </td><td> 시그마 </td><td> 268,054 </td><td> 12.5 μg / ML 작업 희석 </td></tr><tr><td> 나트륨 차아 염소 산염 </td><td> 모든 브랜드 </td><td> 5 % 가정용 표백제 </td><td> 신선한 표백제를 사용하십시오. </td></tr><tr><td> 수산화 나트륨 </td><td> 모든 브랜드 </td><td> CAS 1310-73-2 </td><td> 5 N 주식 </td></tr><tr><td> M9 배지 </td><td> Wormlab 제조법 도서 </td><td> <a href="http://130.15.90.245/wormlab_recipe_book.htm#Commonlab">http://1</a>30.15.90.245/wormlab_recipe_book.htm # Commonlab </td><td> 26 </td></tr><tr><td> levamisol </td><td> 시그마 </td><td> 31,742 </td><td> 100 μm의 - 1 MM 근무 희석 </td></tr><tr><td> 나트륨 azide </td><td> 피셔 과학 </td><td> S227 </td><td> M9 근무 희석 10 MM </td></tr><tr><td> 24 잘 플레이트 </td><td> Greiner 바이오 원 </td><td> 662,160 </td><td> VWR 분배기 </td></tr><tr><td> 현미경 슬라이드 </td><td> 모든 브랜드 </td><td> 75 X 25 X 1mm </td><td></td></tr><tr><td> 현미경 커버 전표 </td><td> 모든 브랜드 </td><td> 22 X 22mm No.1.5 </td><td> 현미경 제조 업체에서 권장하는 두께를 사​​용하십시오. </td></tr><tr><td> 복합 현미경 </td><td> 칼 자이스 혈구 주식 회사 </td><td> A1m </td><td> 실험의 요구에 맞게 목표 및 필터를 사용하십시오. </td></tr><tr><td> 미디어 펌프</td><td> Manostat Varistaltic 펌프 </td><td> 케이트 모델 # 72-620-000 </td><td> 관 그리고 기계에 맞는 설정을 사용 </td></tr></tbody></table>

rnai screening to identify postembryonic phenotypes in c. elegans

C. elegans는 많은 유전자와 유전자 경로 1의 발견과 기능적 특성에 대한 유용한 모델 시스템 것으로 입증되었습니다. 보다 정교한 도구와이 시스템의 연구를위한 자원이 더 미묘한 phenotypes이나 역할과 유전자의 지속적인 발견을 촉진하고 있습니다. 여기에서 우리는 C를 식별 맞게 일반화된 프로토콜을 제시 RNAi 2를 사용 흥미 postembryonic phenotypes와 elegans의 유전자. 이 절차는 쉽게 여부 해부 또는 복합 현미경의 빛 또는 형광 광학에 의해 검정 선택의 표현형으로 수정됩니다. 이 심사 프로토콜은 유기체의 물리적 자산 및 분자 도구 C.에 capitalizes elegans 연구 커뮤니티하였습니다. 예를 들어, 우리는 이것의 일반적인 로컬 라이제이션에 필요한 유전자를 식별하기 위해 RNAi 화면에 형광등 제품을 표현하는 통합 transgene의 사용을 보여줍니다늦은 무대 애벌레와 어른의 제품입니다. 첫째, 우리는 전체 길이 cDNA 삽입으로 상용 게놈 RNAi 라이브러리를 사용했습니다. 이 라이브러리는 후보 유전자 산물의 RNAi 감소하여 여러 후보자의 급속한 식별을 용이하게합니다. 둘째, 우리는 RNAi를 구분 배경에 관심이 우리의 fluorecently 태그가 단백질을 표현 통합 transgene을 올렸습니다. 셋째, RNAi에 부화 동물을 노출하여,이 화면은 특별히 관심 단백질을 규제에서 사후 배아 역할을 숨길 수있는 중요한 배아 역할을 유전자 제품의 식별을 허용합니다. 마지막으로,이 화면은 단세포 결의안에 대한 갖춘 복합 현미경을 사용합니다.

Watch this Scientific Journal Video about RNAi Screening to Identify Postembryonic Phenotypes in C. elegans at JoVE.com

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

우리의 단백질 표현 및 현지화의 postembryonic 규제를 식별할 수 sensitized 방법을 설명<em&gt; C. elegans</em&gt; RNAi 기반 게놈 화면과 기능, fluorescently 태그가 단백질을 표현 통합 transgene을 사용합니다.

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

rnai-screening-to-identify-postembryonic-phenotypes-in-c-elegans

Nanyang Technological University

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RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

17.0K 조회수.  Texas A&M University System Health Science Center. 우리의 단백질 표현 및 현지화의 postembryonic 규제를 식별할 수 sensitized 방법을 설명 C. elegans RNAi 기반 게놈 화면과 기능, fluorescently 태그가 단백질을 표현 통합 transgene을 사용합니다.

비디오: RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

연구

생물학

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

의 정량 및 자동화된 고속 처리량 게놈 전체의 RNAi 화면 C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

에서 Hypoxia의 효과를 연구하기 위해 정의된 기체 환경 만들기<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans의 주 화성 분석

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

항체 염색법에<em&gt; C. 엘레</em&gt; &quot;동결 크래킹 사용&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

대규모 유전자 넉다운에<em&gt; C. 엘레</em&gt; 강력한 기능 상실 표현형을 생성하는 dsRNA를 먹이는 라이브러리 사용

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

허혈 / 재관류에 대한 산소 결핍 - 기아 모델<em&gt; C. 엘레</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

방법은 근육의 세포 내 구획을 평가하기 위해<em&gt; C. 엘레</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...