저자 프로프 감사합니다. 키아 라 캄파넬라과 경쟁력의 암라 센터, 레이저 microdissector의 사용과 그들을 제공하는 나폴리 페데리코 II 대학, 나폴리, 이태리, 그리고 3D 애니메이션 관대한 도움 씨 빈센조 Vicidomini.

1 부. 구체적이고 현지화된 RNAi의 대상이 Drosophila Imaginal 윙 디스크의 준비. 생물 학적 소재로, 우리는 GAL4/UAS 시스템 1에 의해 언어 및 특정 유전자 입을 대상이 유전자 변형 애벌레에서 얻은 Drosophila imaginal 윙 디스크를 사용했습니다. 하나 GAL4 드라이버 UAS 응답 두 번째를 가지고이 애벌레 자손은 두 부모의 라인과 관련된 유전자 십자가에서 orginates. 특정 시간적 및 / 또는 공간적 패턴에 GAL4을 표현하는 것 외에도, 드라이버 라인은 GAL4 표현 도메인을 시각화 수 UAS - GFP 기자를 수행합니다. UAS 응답 라인, UAS 시퀀스의 통제를 받고 특별히 관심을 선택한 유전자 (유전자 X 불러야 2) 타겟팅할 수 RNA (hpRNA)을 입을 헤어핀을 표현. 일단 세포로 표현, hpRNA X는 특별히 선택된 유전자를 침묵 할 수 작은 간섭 RNA (siRNA)를 생성 죽습니다. 애벌레 자손에서, 그 드라이버와 UAS - 입을 구조에만 GAL4 단백질을 표현하는 그 세포 베꼈는데는 응답 transgenes을 모두 실시하고, 따라서 선택된 유전자 X의 공간 제한 입을를 유도 수 있습니다 가능한 응용 프로그램의 다양한 가운데, 우리는 engrailed - GAL4의 통제하에 우리의 관심이 선택한 유전자 (유전자 X) (EN) 드라이버, 날개 디스크 3의 후부 구획에서 특정 입을를 실행할 수있는 침묵이 접근 방식을 적용 , 그의 영토는 UAS - GFP transgene의 표현에 의해 표시되었다. 침묵 imaginal 날개 디스크가 손으로 해부 특히 주변 오염 조직, 준수 tracheas를 제거하기 위해 돌보는했다. 각 절연 날개 디스크 다음 신속하고 부드럽게 선택 영역의 즉각적인 레이저 microdissection을위한 이전 처리, RNase 무료 해부 프레임으로 전달했다. 2 부. 날개 디스크의 침묵 (후부)와 unsilenced (앞쪽에) 구획에서 선택한 영역의 레이저 microdissection. 그리고 unsilenced (앞쪽에, GFP - unlabelled) 구획이 실현되었다;되면 날개 디스크 침묵 (GFP - 표시 후부)에서 특정 영역의 레이저 microdissection은 멤브레인을했다. 이것은 쉽게 모두 들어갈 수있는 영역을 그림으로 수행 되었음 디스크의 GFP - 긍정적이고 부정적인 측면 GFP -. microdissector 아래, 우리는 먼저 선택한 경계선을 잘라 만드는 GFP 양성 세포에 레이저 광선을 하였다. microdissector 선택한 속도와 전력 절감과 함께 진행지만, 조직의 선택된 영역 아래에있는 튜브에 폭포까지 그것은 수동으로 절단 범위를 좁힐 수 있습니다. 이 튜브는 GFP 양성 세포는 이후 RNA 추출을위한 준비가되도록, TRI 시약의 작은 볼륨을 포함하고 있습니다. 우리는 이후 unsilenced, GFP - 부정적인 조직에서 이에 상응하는 지역을 해부하다하기 위해, 날개 디스크의 GFP - 부정적인 측면, 반대로 절단 영역을 옮겼습니다. 다시 한번, 자동 절단 후, 우리는 수동으로 절단을 수정하기 위해 진행. 일단 상처는 RNA 추출을위한 준비 또한 GFP - 부정적인 조직이 TRI 시약에서 발견되었습니다, 완료했다. 부 3. Microdissected 조직 분야에서 RNA 추출 및 전송 프로파일. 우리는 모자에서 액체를 분리하기 위해 튜브를 늘인 다음 표준 TRI 시약 프로토콜 다음 RNA 추출을 수행하는 1 ML에 TRI 시약을 추가했습니다. 충분한 자료를 수집하기 위해, 우리는 100 imaginal 날개 디스크에있는 절단 절차를 반복했다. 5000에 대한 μm의이 각각 100 지역에서 시작, 우리의 수율은 RNA 1.5 μg의되었습니다. 매뉴얼 해부의 정확도는 다음 랜덤 Hexamers와 cDNA 합성하는 슈퍼 스크립트 III (Invitrogen)를 사용하여 RT - PCR에 의해 침묵과 unsilenced 샘플에서 GFP 발현을 선택하여 제어했습니다. 함께 자사의 규제 경로의 putative 대상의 사람과 침묵 유전자 X의 표현 수준을 결정하기 위해 주력하고 이후 정량 분석​​은 마스터 믹스 (Invitrogen)을 사용하여 실시간 RT - PCR에 의해 수행되었다.

<ol>
	<li>Elliott, D. A., Brand, A. H., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GAL4+System+A+Versatile+System+for+the+Expression+of+Gene.">The GAL4 System A Versatile System for the Expression of Gene.</a> , (2008).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.+.">Wing disc development in the fly: the early stages. .</a> Current Opinion in Genetics &#38; Development. 11, 470-475 (2001).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-U1-151-U1 (2007).</li><li>Martin, F. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+the+control+of+growth+in+the+Drosophila+wing+imaginal+disc.">Compartments and the control of growth in the Drosophila wing imaginal disc.</a> Development. 133, 4421-4426 (2006).</li><li>Tabata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+morphogen+gradients.">Genetics of morphogen gradients.</a> Nature Reviews Genetics. 2, 620-630 (2001).</li><li>Moreno, E. &. a. m. p. ;. a. m. p., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dMyc+transforms+cells+into+super-competitors.">dMyc transforms cells into super-competitors.</a> Cell. 117, 117-129 (2004).</li></ol>

unsilenced 날개 디스크의 앞쪽에 / 후부 구획에 달성 자신의 기초 표현 수준과 관련하여, 선택된 유전자 X의 활동이 입을시 40 %까지 감소 발견되었습니다. 대조적으로, 자사의 putative 목표 (유전자 Y) 중 하나는 우리가 부정적인 유전자 X. 규제했다는 가설을 검증하기 위해 선도적인, (7 접이식 증가)까지 - 규제 대폭 발견 우리는 위에서 설명한 실험 방법이 성공적으로 다양한 규?...

변종 플리 Drosophila UAS - 입을 라인 VDRC RNAi 모음 2에서 구할 수 있습니다. GAL4 - 드라이버 라인 컬렉션은 블루밍턴 Drosophila 증권 센터 (인디애나, 미국)에서 사용할 수 있습니다. 장비 필요한 장비는 레이저 microdissector (Leica LMD6000), 쿨터 Microfuge 22R (베크맨), PCR (내 자전거 타는 사람을 BIO - RAD), 실시간 PCR (BIO - RAD iQ5)가 포함되어 있습니다. 도구 필요한 도구는 다음과 같습니다 유리 웰스, 브러시, microdissection의 포셉, 매달려 드롭 슬라이드, 주사기 바늘, PET 멤브레인, 플라스틱 튜브 (50 ML, 05 ML, 0.25 ML)과 금속 프레임에 장착된 곤충 핀합니다.

laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc

조직의 이질적인 성격 스트림 분석을 손상, 생물 학적 샘플에서 생성된 수있는 정보의 양을에 제한 요소로 입증되었습니다. 생체내 상호 작용의 분자 철저한 분석을 요점을 되풀이하기 위해 많은 조직 내의 기존의 복잡하고 역동적인 세포 연결을 고려 하나는 자신의 기본 컨텍스트 내에서 특정 세포 인구를 분석할 수 있어야합니다. 레이저 중재의 microdissection은 분자 무결성을 유지하면서 직접 미세한 시각에 따라 관심의 세포의 모호하지 식별 및 풍작을 수 있도록,이 목표를 달성하실 수 있습니다. 우리는 성장 조절, 세포 분화 및 organogenesis를 연구하는 유리한 모델 시스템을 대표하는 개발 Drosophila 날개 디스크의 정의된 영역 내에서 유전자 발현을 분석하는이 기술을 적용했습니다. 애벌레 imaginal 디스크는 천재적으로 가계 제한 경계에 의해 앞쪽에 및 사후, 지느러미 및 복부 구획으로 세분화됩니다. inducible GAL4 - UAS 이진 표현 시스템이 구획의 각 특별히 만들 GAL4 - 드라이버 해당의 통제하에 UAS - GFP transgene을 표현 형질 전환 파리에서 라벨 수있는 사용하고. 형질 디스크에서 세포의 개별 하위 집합의 유전자 발현 프로 파일링은 정확하게 레이저 절단을 안내하기 위해 형광 GFP 신호를 사용하여 레이저 중재 microdissection 후 결정하실 수 있습니다. 하류 응용 프로그램의 다양한 가운데, 우리는 지역화된 RNA 간섭 (RNAi) 이후 RNA의 성적표 프로 파일링에 집중했다. RNAi 기술의 출현과 함께, GFP 라벨은 특정 유전자 입을 수있는 분리된 세포 인구의 transcriptional 응답을 한정된 수 있도록, 주어진 유전자의 현지 최저와 결합 수 있습니다. 이 방법의 유효성을 검사하기 위해, 우리는 (GFP 표현으로 표시) 뒷부분에서 디스크의 동일한 영역을 해부하고, 그렇지 않으면 ubiquitously 표현 유전자의 P의 구획에 지역 입을시 앞쪽에 (unlabelled) 구획. RNA는 microdissected 침묵과 unsilenced 영역과 정량 실시간 RT - PCR에 의해 결정 비교 유전자 발현 프로 파일링에서 추출한했다. 우리는이 방법이 효과적으로 Drosophila imaginal 디스크 내에서 세포의 하위 집합의 정확한 transcriptomics에 대해 적용할 수 것을 보여줍니다. RNA의 소스로 거대한 디스크 준비가 일반적으로 세포 균질성을지지하면서 실제로, 그것은 잘 transcriptional 표현이 위치 정보의 결과에서 이러한 구조 내에서 크게 달라질 수 있다고 알려져 있습니다. 레이저 절단을 안내화된 형광 GFP 신호를 사용하여보다 정확한 transcriptional 분석이 수행하고 수익성 자신의 기본 컨텍스트 내에서 서로 다른 세포 lineages의 사본 프로 파일링 포함하여 서로 다른 응용 프로그램에 적용할 수 있습니다.

Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

레이저 microdissection은 현지화된 RNAi를 받게 Drosophila 날개 디스크의 특정 구획에있는 유전자 발현 프로 파일링을 분석에 적용되었다<em&gt; 생체내에</em&gt;. 침묵과 unsilenced 구획의 상당 지역에서 추출한 RNA는 기본 조직 microecology의 컨텍스트 내에서 비​​교 유전자 발현 프로 파일링을 결정하는 정량 RT - PCR로 분석했다.

레이저 Microdissection는 세포의 하위 집합 유전자 발현 프로 파일링에 적용 Drosophila 윙 디스크

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

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Biology

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

11.1K 조회수.  University of Naples. 레이저 microdissection은 현지화된 RNAi를 받게 Drosophila 날개 디스크의 특정 구획에있는 유전자 발현 프로 파일링을 분석에 적용되었다 생체내에. 침묵과 unsilenced 구획의 상당 지역에서 추출한 RNA는 기본 조직 microecology의 컨텍스트 내에서 비​​교 유전자 발현 프로 파일링을 결정하는 정량 RT - PCR로 분석했다.

비디오: Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

포유 동물 조직의 레이저 캡처 Microdissection

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

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생물학

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

의 파라핀 - 임베디드 및 냉동 섹션 Drosophila 성인 근육

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

에서 태그가 핵의 선호도 기반 격리<em&gt; 초파리</em유전자 발현 분석을위한&gt; 조직

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.