MEH HJ 반 다니엘 교수 일리노이 대학, 어바나-샴페인에 의해 투자 되었다. 또한, 자금에 대 한 우리 감사 합니다 (M.E.H. T.G.)를 인간 프론티어 과학 프로그램 및 유럽 사회 기금 및 체코 공화국의 국가 예산 프로젝트 아니오. (하 T.G.) CZ.1.07/2.3.00/30.0041입니다. 우리 감사 바다 광학 출판 비용을 커버.

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</ol>

대양 광학이이 원고에 대 한 페이지의 비용을 투자 했다.

계란 제거 실험 무리 기생충-호스트 coevolution66를 공부 하는 가장 일반적인 방법 이지만, 재료, 기술, 또는 프로토콜 표준화에 공동된 노력 부족은. 이것은 특히 메타 분석에 대 한 문제가입니다. 지금까지 호스트 계란 거절의 우리의 지식, 아니 메타-분석 연구67,68, 모방 또는 비 모방으로 간주 됩니다 포함 한 가운데 방법론 불일치에 대...

무리 기생충 다음 그들의 영을 인상 하 고 부모의 케어1,2,3와 관련 된 비용을 지불 수 있습니다 다른 종의 둥지에 있는 그들의 계란을 낳는다. Outwit 호스트 부분 기생충을 감지 하는 기생충 및 sleuthing 편에서 호스트를 속임수의이 행위는 두 배우에 강한 선택 압력을 제공 한다. 새 무리 기생의 경우에 이질적인 기생 계란의 호스트의 인식 선택 호스트 달걀, 모방 하는 기생충에 대 한 호스트 및 기생충4사이 진화 군비 경쟁을 합니다. 가만히 기생 공부 coevolutionary 역학 조사를 위한 모델 시스템 이기 때문에 중요 하 고 야생5의사 결정 이다. 달걀 제거 실험 분야와 생태학6interspecific 상호 작용 조사 하기 위해 사용 하는 중요 한 도구에서 새 무리 기생 공부에 사용 되는 가장 일반적인 방법 중 하나입니다.
계란 제거 실험의 과정, 연구원은 일반적으로 소개 자연 또는 계란을 모델 하 고 표준화 된 기간 동안 이러한 실험 계란에 대 한 호스트의 응답을 평가. 이러한 실험 수 둥지7, 사이 진짜 계란 (에서 변화 하는 모습)를 교환 하거나 염색 또는 그 계란 그림 진짜 계란 (필요에 따라 추가 패턴)와 그들의 원래 둥지8, 반환 또는 모델 생성의 표면 컬러9,10, 크기11및 모양12안보 등 특성 조작. 다양 한 외관의 계란에 호스트 응답 계란 거절 결정13 어떻게 다른 그 달걀14응답 유도 해야 도달 하는 사용 하는 정보 내용에 귀중 한 통찰력을 제공할 수 있습니다. 최적의 수용 임계값 이론15 상태 호스트 실수로 기생 계란 (수락 오류)을 수락 하거나 실수로 자신의 차이 검토 하 여 그들의 자신의 계란 (거부 오류)을 제거의 위험을 균형 해야 합니다 (또는 그 달걀의 내부 템플릿)과 기생 계란. 따라서, 수용 임계값을 넘어 호스트 결정 자극을 참을 수 너무 다르다 존재 합니다. 기생 위험 낮은 때 수용 오류의 위험이 낮습니다 때 기생의 위험이 높습니다. 따라서, 결정 특정 컨텍스트 이며 인식된 위험 변경14,,1617으로 적절 하 게 이동 합니다.
최적의 수용 임계값 이론 호스트 호스트와 기생충 고기에서 연속 변화에 따라 결정의 기초는 가정 합니다. 따라서, 다양 한 기생충 고기 호스트 응답 측정은 얼마나 관용 (자체 phenotypic 변이)를 가진 호스트 인구는 기생 고기의 범위를 설정 하는 데 필요한. 그러나, 거의 모든 이전 연구는 여 지 없이 그 색과 maculation 치료 (예를 들면, 모방/비 모방)에 의존 했습니다. 호스트 껍질 고기 하지 변화, 생물학적으로 실용적인 기대는, 경우에 모든 것 (흉내의 정도)에 직접 비교. 그렇지 않으면, "모방" 달걀 모델 어떻게 비슷한 그것은 호스트 계란 시간과 결과18을 비교할 때 혼란에 잠재적으로 지도할 수 있던 인구 사이 달라질 수 있습니다. 이론 기생 계란과 그들의 자신의14, 반드시 특정 기생 계란 색의 차이 기반으로 결정 하는 해당 호스트를 제안 합니다. 따라서 단일 계란 모델 형식을 사용 하 여지 않습니다 호스트 결정 임계값 또는 차별 능력, 가설을 테스트 하는 이상적인 접근 하지 않으면 그냥 눈에 띄는 차이 (JND이) 계란 모델 유형 및 개별 호스트 달걀 색은 관심의 변수입니다. 이 또한 교환 또는 색상19범위의 자연에 대 한 호스트 응답을 테스트 하려면 자연 계란을 추가 하는 실험적인 연구에 적용 됩니다. 그러나, 이러한 연구 호스트와 기생충 고기에 변화에 대 한 허용, 하는 동안 그들은 특성6, conspecific 계란7을 사용 하는 경우에 특히 발견 자연 변화에 의해 제한 됩니다.
대조적으로, 다양 한 색상의 인공 계란 연구원은 자연 변이의 제약 조건에서 (예를 들어, 그들은 응답 superstimuli20을 조사할 수), 호스트 인식6의 한계를 조사 하도록 허용. 최근 연구는 그림 실험 계란 일치 하 고 껍질9 에 별색21변이의 자연적인 범위를 능가 하도록 설계 하 여 호스트 응답 phenotypic 범위에 걸쳐 측정 하 소설 기법을 사용. 기본 인지 과정을 설명할 수 있는 호스트 응답 따라 그라디언트 색상으로 계란을 공부 수용 임계값15 등 coevolved 흉내4, 이론적인 예측 사이의 지속적인 차이 기반 하기 때문에 특성입니다. 예를 들어 사용 하 여이 접근, Dainson 외. 21 그 껍질 지상 채색 및 자리 채색 색채 대비 높은 경우 미국의 로빈 Turdus migratorius 경향이 계란을 더 강하게 거부 설립. 이 어떻게이 호스트 정보를 처리,이 경우에 구경, 통해 기생 계란을 제거할지 여부를 결정 하에 중요 한 통찰력을 제공 합니다. 페인트 혼합물을 사용자 지정, 연구원은 정확 하 게 조작할 수 실험 달걀의 색과 호스트의 계란, 사이의 유사성 다른 혼동 요인 안보 패턴10, 계란 크기22 및 계란 등을 표준화 하는 동안 모양23.
추가 복제 및 metareplication24 전과 최근 계란 제거 작업의 격려, 그것은 중요 한 과학자 계통 (다른 호스트 종)7,22, 전체 표준화 방법론을 사용 공간 (다른 호스트 인구)7,22,,2526 및 시간 (다른 번 식 계절)7,22,25,26 ,27, 단지 희소 하 게 수행 했다. 표준화 된28 없었던 방법론 나중 artefactual 결과29,30이어질 표시 했다. 이 종이 달걀 제거 실험 연속 변화에 대 한 응답을 검사 하 고 중요 한 방법론 개념의 수를 강조 하는 이러한 유형의 복제 하고자 하는 연구자에 대 한 지침 역할: 제어 둥지의 중요성 선험적 가설, metareplication, pseudoreplication, 그리고 색상 및 스펙트럼 분석. 계란 제거 실험 조류 호스트 기생충 coevolution 분야 지배에 불구 하 고 아니 포괄적인 프로토콜은 아직 존재 한다. 따라서, 이러한 지침 남북 및 내부-실험실 반복성 어떤 가설의 진정한 시험 metareplication, 즉에 공간 계통, 전체 전체 연구를 반복 고 시간24, 귀중 한 자원이 될 것을 수만 의미 해질 때 일관 된 방법29,,3031를 사용 하 여.

<table><tbody><tr><td>Replicator Mini +</td><td>Makerbot</td><td></td><td></td></tr><tr><td>Professional Acrylic Paint Cobalt Turquoise Light</td><td>Winsor &amp;amp; Newton</td><td>28382</td><td></td></tr><tr><td>Professional Acrylic Paint Titanium White</td><td>Winsor &amp;amp; Newton</td><td>28489</td><td></td></tr><tr><td>Professional Acrylic Paint Cobalt Green</td><td>Winsor &amp;amp; Newton</td><td>28381</td><td></td></tr><tr><td>Professional Acrylic Paint Cobalt Turquoise</td><td>Winsor &amp;amp; Newton</td><td>28449</td><td></td></tr><tr><td>Professional Acrylic Paint Burnt Umber</td><td>Winsor &amp;amp; Newton</td><td>28433</td><td></td></tr><tr><td>Professional Acrylic Paint Red Iron Oxide</td><td>Winsor &amp;amp; Newton</td><td>28486</td><td></td></tr><tr><td>Professional Acrylic Paint Cadmium Orange</td><td>Winsor &amp;amp; Newton</td><td>28437</td><td></td></tr><tr><td>Professional Acrylic Paint Raw Umber Light</td><td>Winsor &amp;amp; Newton</td><td>28391</td><td></td></tr><tr><td>Professional Acrylic Paint Yellow Ochre</td><td>Winsor &amp;amp; Newton</td><td>28491</td><td></td></tr><tr><td>Professional Acrylic Paint Mars Black</td><td>Winsor &amp;amp; Newton</td><td>28460</td><td></td></tr><tr><td>Paint Brush</td><td>Utrecht</td><td>206-FB</td><td>Filbert brush</td></tr><tr><td>Paint Brush</td><td>Utrecht</td><td>206-F</td><td>Flat brush</td></tr><tr><td>Hair Dryer</td><td>Oster</td><td>202</td><td></td></tr><tr><td>Fiber optic cables</td><td>Ocean Optics Inc.</td><td>OCF-103813</td><td>1 m custom bifurcating fiber optic assembly with blue zip tube (PVDF), 3.8mm nominal OD jeacketing and 2 legs</td></tr><tr><td>Spectrometer</td><td>Ocean Optics Inc.</td><td>Jaz</td><td>Spectrometer unit with a 50 um slit width, installed with a 200-850 nm detector (DET2B-200-850), and grating option # 2.</td></tr><tr><td>Battery and SD card module for spectrometer</td><td>Ocean Optics Inc.</td><td>Jaz-B</td><td></td></tr><tr><td>Light source</td><td>Ocean Optics Inc.</td><td>Jaz-PX</td><td>A pulsed xenon light source</td></tr><tr><td>White standard</td><td>Ocean Optics Inc.</td><td>WS-1-SL</td><td>made from Spectralon</td></tr><tr><td>OHAUS Adventurer Pro Scale</td><td>OHAUS</td><td>AV114C</td><td>A precision microbalance</td></tr><tr><td>Gemini-20 portable scale</td><td>AWS</td><td>Gemini-20</td><td>A standard scale</td></tr><tr><td>Empty Aluminum Paint Tubes (22 ml)</td><td>Creative Mark</td><td>NA</td><td></td></tr><tr><td>Telescopic mirror</td><td>SE</td><td>8014TM</td><td></td></tr><tr><td>GPS</td><td>Garmin</td><td>Oregon 600</td><td></td></tr><tr><td>220-grit sandpaper</td><td>3M</td><td>21220-SBP-15</td><td>very fine sandpaper</td></tr><tr><td>400-grit sandpaper</td><td>3M</td><td>20400-SBP-5</td><td>very fine sandpaper</td></tr><tr><td>color analysis software: &amp;lsquo;pavo&amp;rsquo;, an R package</td><td>for use in, &lt;em&gt;R: A language and environment for statistical computing&lt;/em&gt;</td><td>v 1.3.1</td><td>https://cran.r-project.org/web/packages/pavo/index.html</td></tr><tr><td>UV clear transparent</td><td>Flock off!</td><td>UV-001</td><td>A transparent ultraviolet paint</td></tr><tr><td>Plastic sandwich bags</td><td>Ziploc</td><td></td><td>Regular plastic sandwich bags from Ziploc that can be purchased at the supermarket.</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34120</td><td>11 x 21 cm kimwipes</td></tr><tr><td>Toothbrush</td><td>Colgate</td><td></td><td>Toothbrush</td></tr></tbody></table>

probing the limits of egg recognition using egg rejection experiments along phenotypic gradients

무리 기생충 알을 그들의 다른 여자의 둥지에 해치 고 그들의 영을 후방 호스트 부모. 공부 어떻게 가만히 기생충 그들의 영을 양육으로 호스트를 조작 하 고 호스트 기생을 검출 하는 방법 coevolutionary 생물학의 분야에서 중요 한 통찰력을 제공 합니다. 무리 기생충, 뻐꾸기 등 cowbirds, 그들은 그들의 자신의 영을 양육의 비용을 지불할 필요가 없기 때문에 진화 이점을 얻을. 그러나, 이러한 비용 기생충, 계란, 그들의 영, 그리고 성인을 포함 하 여 모든 발달 단계에 대 한 호스트 방어에 대 한 선택 합니다. 달걀 제거 실험 호스트 방어를 공부 하는 데 사용 하는 가장 일반적인 방법은 있습니다. 이 실험 동안 연구원 호스트 둥지에 실험적인 계란을 배치 하 고 모니터링 호스트 응답 하는 방법. 색상은 종종 조작, 그리고 기대 그 차별과 호스트와 실험에 그 간의 호환성의 학위의 가능성은 긍정적으로 관련. 이 종이 같은 실험의 결과 분석을 일관 된 계란 색을 만드는 방법을 설명에서 계란 제거 실험에 대 한 가이드로 제공 합니다. 특별 한 주의 잠재력이 호스트 인식에 색깔 편견을 탐구 하는 새로운 방법을 색상 그라디언트 따라 고유 색된 계란을 포함에 주어 집니다. 표준화, 없이 그것은 결과 의미 있는 방식으로; 연구 사이 비교할 수 이 필드는 표준 프로토콜 추가 실험에 대 한 점점 더 정확 하 고 비교 결과 대 한 수 있습니다.

화려한 달걀 모델 생성
사용자 정의 페인트 혼합물과 자연 계란의 반사율 스펙트럼은 그림 1A-D에 표시 됩니다. 가만히 기생 연구에 사용 되는 페인트 혼합물 자연 반사율 측정 스펙트럼 모양 (색) 및 크기 (밝기)와 밀접 하 게 일치 해야 합니다. 달성 하는 경우 자연 계란 색으로 ?...

Watch this Scientific Journal Video about Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients at JoVE.com

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Phenotypic 그라디언트 따라 계란 제거 실험을 사용 하 여 그 인식의 한계를 조사

이 프로토콜은 계란 제거 실험을 실행 하기 위한 지침을 제공 합니다: 실험적인 그림에 대 한 개요 기술 계란 천연 조류 계란, 현장, 실시 하 고 수집 된 데이터 분석의 색상을 흉내 낸 모델. 이 프로토콜 비교 계란 제거 실험을 실시 하기 위한 획 일 한 방법을 제공 한다.

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate ...

probing-limits-egg-recognition-using-egg-rejection-experiments-along

Research

JoVE Journal

Behavior

8.2K 조회수.  Long Island University-Post. 이 프로토콜은 계란 제거 실험을 실행 하기 위한 지침을 제공 합니다: 실험적인 그림에 대 한 개요 기술 계란 천연 조류 계란, 현장, 실시 하 고 수집 된 데이터 분석의 색상을 흉내 낸 모델. 이 프로토콜 비교 계란 제거 실험을 실시 하기 위한 획 일 한 방법을 제공 한다.

비디오: Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

의 수직 이미지<em&gt; 초파리</em&gt; 계란 챔버

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

연구

발생학

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

공 촛점 분석을 사용하여<em&gt; 아프리카 발톱 개구리 laevis의</em&gt;의 Wnt 및 쉬 Morphogen 그라디언트의 변조기를 조사하기 위해

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

를 사용하여 기능성 복제<em&gt; Xenopus의</em&gt; 난자 발현 시스템

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

완두콩 진딧물 태아의 면역 염색을위한 중요한 조건의 확인 : 조직 투과성을 증가 및 배경 염색을 감소

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

그릴루스 바이마쿨라투스 계란 주입

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Embryogenesis 동안 중복 기능을 테스트하기 위해 역방향 유전자 접근

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

생물학

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

높은 처리량 분석은 개인의 알을 낳는 환경 설정을 검사합니다<em&gt; 노랑 초파리</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

신경과학

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

초파리 애벌레의 열 환경 설정을 결정 하는 온도 분석 결과

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Drosophila Egg Collection and Dechorionation: A Method to Remove the Outermost Egg Layer

Source: Koyle, M. L., et al. <a href="https://www.jove.com/video/54219/" target="_blank">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. J. Vis. Exp. (2016).
The Drosophila embryo is surrounded by protective outer membranes. To gain access to the embryo, the chorion&#8211;the outermost of the membranes&#8211;is often removed. This video describes a commonly used method of dechorionation using sodium hypochlorite. The example protocol demonstrates the procedure while employing sterile techniques necessary for axenic or gnotobiotic animal rearing.

초파리 난자 채취 및 탈echorionation: 가장 바깥쪽 난자를 제거하는 방법

Source: Koyle, M. L., et al. Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions. J. Vis. Exp. (2016).
The Drosophila ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Embryo

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Egg Laying Assay: 예쁜꼬마선충의 알을 낳는 행동을 정량화하는 방법

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

Phenotypic 그라디언트 따라 계란 제거 실험을 사용 하 여 그 인식의 한계를 조사

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