Name: quantifying abdominal pigmentation in drosophila melanogaster
Uploaded: 2017-06-01T12:30:00
Description: Watch this Scientific Journal Video about Quantifying Abdominal Pigmentation in Drosophila melanogaster at JoVE.com

이 작품은 National Science Foundation에서 IOS-1256565 및 IOS-1557638을 AWS에 기부하여 지원되었습니다. 우리는 Patricia Wittkopp과 3 명의 익명의 평론가들에게이 백서의 이전 버전에 대한 도움이되는 의견에 감사드립니다.

1. 시편 설치 참고 : 이미징하기 전에 죽은 파리를 물에 70 % 에탄올에 보관하십시오. <ol><li> 60 mm x 15 mm 페트리 접시에 끓는 물에 용해 된 1.25 % 한천 10 ML을 부어 그것을 설정할 수 있습니다. </li><li> 해부 현미경, 젤의 표면에 ~ 20mm 옹이, 2mm 폭, 1mm 깊이 홈을 만들기 위해 미세 포인트 포셉 한 켤레를 사용합니다. 미세 포셉을 사용하여 성인 파리의 복부 측면을 그루브에 삽입하?...

이 방법론은 다중 하류 분석에 적합한 양적 형태로 색소 침착 데이터를 정확하고 신속하며 반복적으로 수집 할 수있게합니다. 이 방법은 파리의 등선에서 복부 색소 침착에 대한 온도 효과에 대한 자료를 수집하는 데 사용되었습니다. 그러나 전향 적 유전학 연구에서 개인, 개체군 또는 종 사이의 색소의 차이를 결정하는 유전자를 식별하거나 특정 유전자의 색소 침착 패턴에 미치는 영향을 조사...

색소 침착은 종, 개체군, 개개인간에, 심지어 개체 발생 1 , 2 , 3 , 4 , 5 , 6 동안 개인 내에서도 거대한 표현형의 변화를 보여줍니다. 다양한 동물에서 색소 침착에 대한 수많은 연구가 있지만 염색은 Drosophila melanogaster에서 가장 잘 연구되었을 것입니다. 분자 유전학의 모든 힘이 색소 침착을 조절하는 발달 및 생리적 메커니즘을 밝혀 내고 이러한 메커니즘이 어떻게 발전하는지 1 , 6 . D. melanogaster 7 과 8 에서 색소의 생화학 적 합성을 조절하는 유전자와 시간 및 공간적 di를 조절하는 유전자에 대해 많이 알려져있다 .이 생합성에 대한 스트라이핑 9 , 10 , 11 , 12 , 13 . 또한, 유전지도 작성은 D. melanogaster 14 , 15 , 16 , 17 에서 착색의 내부 및 내부 특이적인 차이점을 나타내는 유전자 좌의 위치를 ​​확인했다. 행동 18 , 19 , 면역 19 , 20 과 같은 착색과 pleiotropic 특성 사이의 관계도 착색 패턴 15 , 21 , 22 의 적응 의미를 가지고 탐험되었습니다. 이와 같이, D. melanogaster의 색소 침착 은 강력하면서도 단순한 m복잡한 표현형의 개발과 진화를위한 odel. 성인에서 색소 침착 D. melanogaster 는 특히 날개와 지느러미 흉부와 복부에서 몸 전체에 뚜렷한 무늬가 특징입니다. 그러나 대부분의 연구 관심을받은 것은 배 쪽 복부에있는 각 큐티 큘라 플레이트 (tergite)의 착색입니다. 유전 적 17 , 23 및 환경 24 , 25 요인 때문에이 색소 침착 ( 그림 1A- F )에 상당한 차이가 있습니다. 복강 각질의 큐티클은 전방 및 후방 발달 구획으로 구성되며 ( 그림 1G ), 각 구별은 색소 침착 및 장식에 따라 더 세분화 될 수 있습니다. 전방 구획에는 6 개의 큐티클유형 (a1-a6)을 포함하고 후방 구획은 세 가지 (p1-p3)를 포함한다 ( 그림 1G ). 이 중 p1, p2 및 a1 표피는 일반적으로 미연 신 (un-stretched abdomens)의 표층 아래에 ​​접혀있어 숨겨져 있습니다. 확실하게 눈에 보이는 표피는 큐티클 유형 a4 (중간 정도의 털이있는 털이)와 a5 (털이 큰 털이있는)로 구성되어 있고 밴드의 뒷부분 가장자리로 구성된 무거운 색소의 밴드를 특징으로합니다. ( 그림 1G )보다 강하게 색소 침착되었다. 이 밴드의 앞부분은 가볍게 착색 된 털이 많은 큐티클의 영역으로, 뒤쪽으로 (a3) ​​딱딱하고 앞쪽으로는 (a2)가 없습니다. 파리 사이의 색소 침착의 변이는 색소의 강도와 색소의 폭 모두에서 관찰된다. 일반적으로, 변이는 대부분의 후부 분절 (복부 분절 5, 6 및 7)에서 가장 크며, 더 많은 전 안부 분절에서 더 낮다 (복부3과 4 장) 24 . 또한, D. melanogaster 색소 침착에 성적 이형성이 있으며, 남성은 일반적으로 5 번째 및 6 번째 복부 tergites를 완전히 색칠합니다 ( 그림 4C ). D. melanogaster 의 복부 색소 침착에 대한 대부분의 연구에서, 색소 침착은 14 , 15 , 16 , 17 , 24 , 30 의 척도에서 정 성적으로 27 , 28 , 29 또는 semi-quantitatively로 측정 된 패턴을 가진 범주 형 또는 서수 형으로 처리되었습니다. , 31 , 32 , 33 , 34 , 35, 36 , 37 . 이러한 방법은 필연적으로 정밀도 부족으로 고통 받고 있으며 색소 침착의 주관적인 평가에 의존하기 때문에 여러 연구에서 데이터를 비교하기가 어렵습니다. 몇몇 저자들은 착색 38,39의 공간적 차원, 특정 큐티클 유형 23 , 25 , 39 , 40 의 착색 강도 또는 복부 용제 전체의 착색 강도 평균을 41 , 42 , 43 로 계량화했습니다. 그럼에도 불구하고 이러한 정량화 방법은 복부 색소 침착의 강도와 공간 분포를 동시에 측정하지 않으므로 색소 침착이 abd에서 어떻게 변하는 지에 대한 뉘앙스를 포착하지 않습니다ominal tergite. 또한, 이러한 정량화 방법들 (38 , 41 , 42 , 43) 중 몇 가지는 복부 표피의 해부 및 장착을 필요로한다. 이것은 시간이 많이 걸리고 샘플을 파괴하므로 추가적인 형태 학적 분석에 사용할 수 없습니다. 복부 색소 침착의 발달과 진화에 대한 이해가 심화됨에 따라 공간 분포와 색소 침착을 신속하고 정확하게 측정 할 수있는보다 정교한 도구가 필요합니다. 이 방법의 전반적인 목표는 D. melanogaster 에서 복부 색소 침착에 대한 복제 가능하고 정확한 측정을 얻기 위해 디지털 이미지 분석을 이용하는 것 입니다. 이 방법론은 세 단계로 구성됩니다. 첫째, 성충은 비파괴 적으로 장착되고 등 쪽 복부의 디지털 이미지가 촬영됩니다. 둘째, ImageJ 매크로를 사용하여 사용자는 a2 큐티클의 앞쪽에서 세 번째 및 네 번째 복부 세그먼트의 a5 큐티클 (녹색 상자, 그림 1G )의 후방까지 연장되는 픽셀의 앞 / 뒤쪽 스트립을 정의합니다. 이 스트립의 너비에 걸친 평균 픽셀 값은 장축을 따라 추출되어 테르가 트의 전방에서 후방으로 변화함에 따라 색소의 공간 분포와 강도를 포착하는 프로파일을 생성합니다. 셋째, R 스크립트는 큐빅 스플라인을 사용하여 수학적으로 착색 프로파일을 설명하는 데 사용됩니다. 그런 다음 R 스크립트는 스플라인과 첫 번째 및 두 번째 미분을 사용하여 a2-a5 표피의 너비, 안료 밴드의 너비 및 염색의 최대 및 최소 레벨을 추출합니다. 따라서이 방법은 복부 색소 침착의 깊이와 공간 특성을 정량화합니다. 이 방법론은 세 번째 및 네 번째 복부 tergites의 착색을 정량화,이들은 이전의 많은 연구 1 , 15 , 23 , 24 , 25 , 28 , 33 , 39 , 42의 초점이었으며, 오직 더 많은 후부 표적과 배타적으로 또는 조합으로 사용되었다. 다섯 번째 및 여섯 번째 복부 tergites보다 덜 변하지 만, 세 번째 및 네 번째 tergites은 남성에서 완전히 색칠되지 않습니다, 그래서이 프로토콜은 남성과 여성 모두에 적용 할 수 있습니다. 그럼에도 불구하고, 여기에 표시된대로, 프로토콜은 여성의 다섯 번째와 여섯 번째 복부 tergites의 색소를 측정하는 데 사용할 수 있습니다. 또한 염색 프로파일의 특성을 추출하는 데 사용되는 스크립트의 사소한 수정을 통해 다양한 방법으로 색소 침착의 변이를 정량화하는 데 사용할 수 있습니다유기체.

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			<td>Dumont #5 Biology Forceps</td>
			<td>FST</td>
			<td>11252-30</td>
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			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>5040</td>
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			<td>Dissecting Scope</td>
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			<td>Leica</td>
			<td>DFC280</td>
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			<td>Gooseneck Cold Light Source</td>
			<td>Schott</td>
			<td>ACE 1</td>
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			<td>Image Acquisition Control Software</td>
			<td>Micro-Manager v1.3.20</td>
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			<td>https://micro-manager.org/</td>
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			<td>Image Analysis Software</td>
			<td>ImageJ</td>
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			<td>Data Analysis Software</td>
			<td>R 3.3.2</td>
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			<td>https://www.r-project.org/</td>
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			<td>LED</td>
			<td>Thor Labs</td>
			<td>LEDWE-15</td>
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			<td>Multimeter</td>
			<td>Fluke</td>
			<td>Fluke 75 Series II</td>
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			<td>60 x 15 mm Petri dish</td>
			<td>Celltreat Scientific Products</td>
			<td>229663</td>
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			<td>Stage micrometer</td>
			<td>Klarman Rulings, Inc.</td>
			<td>KR-867</td>
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quantifying abdominal pigmentation in drosophila melanogaster

색소 침착은 형태 적으로 간단하지만 매우 가변적 인 특성으로 종종 적응의 중요성이 있습니다. 그것은 형태 학적 표현형의 발달과 진화를 이해하기위한 모델로서 광범위하게 사용되어왔다. Drosophila melanogaster의 복부 색소 침착 은 특히 ​​유용하여 연구원은 형태학의 내부 및 내부 특이성 변이의 기초가되는 좌위를 확인할 수있었습니다. 그러나, D. melanogaster 복부 색소 침착은 색소 침착 데이터에 적용 할 수있는 통계 분석의 형태를 제한하는 정량적 인 것이 아니라 정량적으로 정량적으로 분석되어왔다. 이 작품은 성인 D. melanogaster 의 복부 색소 침착 패턴의 다양한 측면을 정량화 할 수있는 새로운 방법론을 설명 합니다. 이 프로토콜에는 표본 장착, 이미지 캡처, 데이터 추출 및 분석이 포함됩니다. 이미지 캡처 및 분석 기능 매크로에 사용되는 모든 소프트웨어오픈 소스 이미지 분석을 위해 작성되었습니다. 이 방법의 장점은 다른 이미징 시스템에서 재현성이 높은 방법론을 사용하여 색소 특성을 정확하게 측정 할 수 있다는 것입니다. 이 기법은 성인 D. melanogaster 의 tergal pigmentation 패턴의 변이를 측정하는 데 사용 되었지만 ,이 방법은 무수히 많은 유기체에서 색소 침착 패턴에 유연하고 광범위하게 적용될 수 있습니다.

프로토콜은 복부 색소 침착에 온도를 키우는 효과를 탐색하는 데 사용되었습니다. 이전 연구에 의하면 발달 온도의 증가는 D. melanogaster 30 , 32를 포함 하여 여러 종의 초파리 에서 복부 색소 침착의 확산을 감소시키는 것으로 나타났습니다. 특히, 복부 tergites 3과 4, 착색의 범위 (안료 밴드의 폭) 17 ° C에서 25 ° C로 ?...

Watch this Scientific Journal Video about Quantifying Abdominal Pigmentation in Drosophila melanogaster at JoVE.com

Quantifying Abdominal Pigmentation in Drosophila melanogaster

이 작품은 디지털 이미지 분석을 사용하여 Drosophila melanogaster의 복부 색소 침착 을 빠르고 정확하게 정량화하는 방법을 제시합니다. 이 방법은 표현형 획득과 데이터 분석 사이의 절차를 합리화하고 표본 장착, 이미지 수집, 픽셀 값 추출 및 특성 측정을 포함합니다.

복부 색소 침착 정량화<em&gt; Drosophila melanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

The overall goal of this methodology is to rapidly quantify abdominal pigmentation in the fruit fly Drosophila melanogaster. This method can help answer key questions in the field of evolutionary biology such as identifying the genetic loci that underlie in turn in trust-specific variation morphology and understanding the development and evolution of morphological phenotypes. The main advantage of this technique is that it allows users to non-destructively measure various aspects of abdominal pigmentation including the spread and depth of pigmentation.Though this method can provide insight into Drosophila melanogaster's abdominal pigmentation, it can also be applied to other Drosophila species and easily adapted to measure pigmentation patterns in other insects and animal taxa. Prior to imaging, the flies can be stored in 70%ethanol to maintain their pigmentation. Visualization requires a mounting pad.Load a 60 millimeter Petri dish with about 10 milliliters of 1.25%agar in water. Under a microscope, use fine forceps to dig a 20 millimeter long, two millimeter wide, one millimeter deep trench into the gel. Several trenches can be made in one dish.Then embed flies into the trench with the surface for analysis sitting just above the gel surface. In this case, the dorsal side is of interest. Now, fill the dish with 70%ethanol until the flies are completely submerged.This reduces reflections off the cuticle that can obscure the pigmentation pattern. Now, start the imaging software and digital camera connected to the microscope. For a light source, cold light from a double gooseneck device is ample.In the image analysis software, set the image type to 8-bit. Then in the image capture software, toggle the Live setting to open a view window of the camera. Set the image type to Grayscale.Now, set the light source to its maximum intensity and position the lights about 120 millimeters from the stage. Set the microscope to 60 times magnification so the field of view is three millimeters wide. Then place the two millimeter stage micrometer into the field of view and focus on it.Returning to the software, adjust the exposure time to view the micrometer using the field found in the exposure box. Then set the scale. Open the straight line tool and draw the length of two millimeters along the stage micrometer.Then input that length under the Set Scale option. The window should then display the scale in pixels per micron. Take note of the scale and OK the adjustments.Now, return to the image capture software and use the Stop and Snap commands to capture an image of the stage micrometer. Then return to the image analysis software and save the image as a Tiff file. Now, focus on the dorsal midline of a mounted fly.The midline should be positioned straight up for the best imaging results. Push aside the wings and any other obtruding structures and adjust the lighting to best illuminate the cuticle. Next, set the exposure in the image capture software using the pixel value histogram as a guide to capture the full pixel range of the image without saturation, thereby maximizing contrast.Retain this exposure and lighting throughout the experiment. Now, before capturing an image, ensure that the abdominal segments of interest are in view. Then capture an image and save the image with the sample ID number in the filename.Use the format described in the written protocol. This analysis makes use of a macro provided by the authors. The image series to analysis should all be contained within the same file folder.Now, start the macro and follow the prompted commands. First, identify the location of the stored files. Then identify the location for where the analysis data should be stored.Next, input the number of characters that were used in the filenames so the software reads the image series correctly. The next prompt is for the width of the region of interest. Here, input the number of pixels along the anterior-posterior strip that needs to be analyzed.Now, at the prompt, make a choice to analyze the first image of the series. The other options are to analyze the next image in the series or to exit the analysis. The line tool gets automatically selected.Use it to identify the midline on the image by drawing a line along it in the anterior to posterior direction. Next, follow the macro prompts to identify the posterior edge of Tergite4 using the line tool. The line tool is made available to draw the line from the posterior midline edge to the right lateral edge so that the center of the line is just posterior to the posterior edge of the pigment band.Then define the anterior edge of Tergite4 using the line tool. Draw a line from the anterior midline edge to the right lateral edge. The middle of the line defined by a white square should sit at the anterior edge of the lightly pigmented cuticle.With this information, the macro now generates a pigmentation profile. However, the profile might be inaccurate if bristles or other blemishes cross the strip along which the profile is measured. To correct for this, select the Live preview on the histogram window and adjust the line such that the profile is smooth.For the image analysis strip to accurately capture the various aspects of pigmentation, it is vital to identify an analysis region that will have minimal noise caused by image or specimen irregularities. Now proceed to analyze Tergite3 in the same image and then move on to the next image. The described procedure was used to explore a known change in abdominal pigmentation caused by rearing temperature.The third and fourth tergites were examined several times under various lightings and exposures. Various aspects of the pigmentation were explored. And specifically, it was found that the pigmentation bands were wider when flies were reared at cooler temperatures.The calculated values were then compared to general subjective measures made on the same flies by five different observers. In the assessment of 45 female flies, the average observed ranking of the width of the fourth abdominal pigmentation band was tightly correlated with the macro's automated measurement. The same pigmentation band was also manually measured using imaging software.These results were also strongly correlated with measurements generated using the macro. The software was then tested to measure pigmentation in female ebony mutants which are well-known to have increased pigmentation in the third and fourth pigment bands. The method was also used to measure the fifth and sixth abdominal bands of wild type females.The automated technique always provided useful measurements of the pigment density and band size. After watching this video, you should have a good understanding of how to quickly and precisely quantify abdominal pigmentation in Drosophila. Mastering the technique mostly requires the depth use of forceps.Once mastered, the imaging portion of this technique can be done at a rate of 50 specimens per hour. Extracting the pigmentation measures from these images can be done at a pace of 70 images per hour.

quantifying-abdominal-pigmentation-in-drosophila-melanogaster

Research

JoVE Journal

Genetics

Quantifying Abdominal Pigmentation in Drosophila melanogaster

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

에듀 염색에 의한 초파리 성인 난소에서 미토콘드리아 DNA 복제의 제자리 라벨에

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

연구

유전학

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

높은 지방 다이어트 먹이 초파리 Melanogaster 의 높은 처리량 Triacylglyceride 분석 결과

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

초파리 melanogaster 회전 운동 정량화 시스템 (REQS)를 사용 하 여 운동 수준 측정

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

초파리 melanogaster 운동을 부드럽게 유도 위한 TreadWheel: 간격 훈련 프로토콜

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Drosophila 시각 시스템에서 낮은 풍부한 셀의 정화

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

박테리아에서 단백질 RNA 결합을 정량화하기위한 분석

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Drosophila를 사용하여 질병 관련 희소한 인간 이체의 생체 내 기능적인 연구

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

소독멜라노가스터에서 새로운 신경보호 유전자를 식별하기 위한 생체 전방 유전자 스크린

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5&#8217;UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.

깨지기 쉬운 X 정신 지체-1 유전자에서 시토신 구아닌-구아닌 트리뉴클레오티드 반복을 정량화하기 위한 견고한 폴리머라제 연쇄 반응 기반 분석

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.