우리는 티가과 성장 감각 예측의 시간 경과 영화를 제공하는 유전자에 대한 구조를 생성하는 그의 도움 마이클 베이커 감사드립니다. 우리는 또한 공압 총기 실험과 기술 지원 버지니아 Vandelinder 감사하고 싶습니다.

입자는 이러한 dsRNA 분자, DNA의 plasmids 또는 염료와 같은 다른 시약과 코팅 수 있습니다. 입자의 종류와 직경이 시약과 조직의 목표 세포의 깊이에 따라 선택됩니다 더욱 침투 큰 입자, 그러나 조직에 손상을 일으킬 수 있습니다. 1. dsRNA 코팅 입자의 준비 <ol><li> 얼음 이소프로판올 100 %를 놓으십시오. </li><li> 100 ML 바인딩 버퍼에, Resuspend 5 MG 골드 입자 (평균 직경 1.6 μm의 S1600ri, 시쉘 기술, LLC.) 솔루션 준비가되면, 다음 단계로 진행합니다. </li><li> 바인딩 버퍼, 소용돌이 간략와 clumping 피하기 위해 1-2 분 sonicate 100 μl를 추가하여 입자 솔루션을 희석. </li><li> dsRNA / siRNA 5 - 10μg (dsRNAs / siRNA가 ~ 1μg/μl의 농도로 물에 용해한다)을 추가합니다. </li><li> 2 분 실온에서 와동과 둡니다. siRNA에 이러한 집중과 시간 간격 결과는 금 입자를 포화. </li><li> 펠렛 ~ 15 초에 대한 벤치 최고 원심 분리기에 ~ 2,500 rpm으로 원심 분리하여 siRNA / 골드 입자 단지. </li><li> 뜨는을 제거하고 부드럽게 펠렛의 최소한의 중단과 함께 750 ML 추위 이소프로판올를 추가합니다. 간략 스핀과 표면에 뜨는를 제거합니다. </li><li> 100 μl 추위 백퍼센트 이소프로판올에서 금 입자를 Resuspend 다음 입자 agglomerates을 깨고 (2-3 펄스) 목욕탕 sonicator에 잠시 sonicate. 마지막으로, 유리 슬라이드에 정지 입자를 기름을 붓고 실온에서 건조하실 수 있습니다. </li></ol> 2. 특정 분석에 대한 실험 설치 준비 <ol><li> 각각의 총기는 최대 ~ 3mm로 ~ 50μm 최소 이르는, 다른 노즐 조리개 직경이 있습니다. 총을 사용하는 특정 따라서 대상으로 조직의 영역에 따라 선택됩니다. </li><li> 그는 압력은 일반적으로 최대 ~ 100μm (높은 압력이 깊은 입자를 제공할 수)에, 대상 세포의 조직에 깊이에 따라 조정됩니다. 배아와 노즐의 끝 사이의 거리는 공기 금 입자 느슨한 기세로 침투 깊이에 영향을 미칩니다. </li></ol> 3. 미리 코팅 입자로 총알을 장전 입자가 매니폴드에 연결된 tygon 튜빙 입자 주입 라인에 건조 분말로로드해야합니다. 4 건조한 분위기에 보관한다이 튜브, ° C 사용하지 않을 같은 시약과 추가 실험에서 다시 사용할 수있을 때. 우리 실험 설정은 다기관에서 별도의 포트를 통해 분석마다 서로 다른 두 가지 시약까지 제공할 수 있습니다. <ol><li> tygon 튜빙 라인에 그들을 로딩하기 전에 유리 커버 슬립이나 면도날의 가장자리를 사용하여 슬라이드 유리에서 건조 코팅 입자를 다쳤어요. 이 tygon 튜빙은 교차 오염을 방지하기는이 시약에 특정한 것입니다. </li><li> 당신이 입자를로드로 전체 관 그리고 집중 가까운 커넥터에 따라 확산 입자를 유지하기 위해 커넥터에 가까운 U 모양으로 튜빙을 구부. 작은 주걱이나 접힌 무게 종이로 입자를 들고, 그리고 태아의 각 시리즈를위한 튜브에 그들의 약 절반을로드합니다. 균일 입자를 확산하기 위해 로딩 단계 동안 튜빙을 누릅니다. </li></ol> 4. 동물 준비 골드 입자 전달 전후의 배아는 실온에서 무균 인공 연못 물에 보관됩니다. <ol><li> 실리콘 고무 (Sylgard 184, 다우 코닝, 미들랜드, MI)의 평면, 5mm 두께의 조각에 새겨진 홈에, 최대 배아, 복부 측면을 놓고 (무균 인공 연못 물에 8 %의 에탄올의 솔루션을 마취시키다 단계 E6 - E8에서 젊은 배아)은 실험을하는 동안 노 에탄올과 인공 연못 물에 남아있을 수 있습니다. </li><li> 즉시 입자를 촬영하기 전에이 솔루션의 일부를 제거하여 태아의 복부 표면을 밝히기 위해서 목욕 수준을 낮추십시오. 그것을 안정화하고 건조로부터 표면을 유지하는 그것의 상단에있는 중간에 잘라 작은 구멍과 함께 조직 종이를 놓습니다. </li></ol> 5. 대상 조직으로 코팅 입자의 딜리버리 입자 중 하나는 부하는 일반적으로 하나의 촬영은 보통 몇 백 입자의 순서에 전달과 함께 최대 10 발이에 사용할 수 있습니다. <ol><li> 총에 해당 튜브 라인에서 입자의 볼러스의 주입을 초래 0.3 s에 대한 밸브 중 하나를 열어 주사를 생성합니다. </li><li> 튜브의 벽에서 입자를 이동시키다하기 때문에 총을의 그 흐름에 자신의 주사를 촉진하는 장면 사이에 부드럽게 튜빙을 누릅니다. </li><li> 관심 조직의 영역을 커버하기 위해 여러 장면이 필요할 수 있습니다. </li><li> 입자 배달 후 다중 잘 플레이트의 인공 연못의 물, 잘마다 선호 하나 배아로 배아를 다시 넣으십시오. </li></ol> 6. 단백질얼룩과의 연결 라벨 RNAi (또는 이소성 표현) 달성 때까지 입자 전달 후 1~3일에 대한 실온에서와 어둠의 배아 보관하십시오. 우리는 다음 실험 표본 및 컨트롤에서 다음 절차 중 하나를 수행 : <ol><li> Netrin의 mRNA의 존재 또는 부재에 대한 분석으로 원위치 하이브리드화에. Digoxigenin - 표시 리치 netrin의 riboprobes는 전체 리치 배아에 hybridized입니다. </li><li> Netrin 단백질과의 연결 조직 (예 : 안티 acetylated tubulin)에 고유한 다른 단백질에 대한 분석을 Immunocytochemistry. 절차는 전체 마운트 배아에서도 수행됩니다. </li><li> 치료 및 제어 분야에 대한 관심의 뉴런 (우리의 경우 mechanosensory 압력 뉴런)의 전체 아보스 레이블을하는 염료는 - 채웁니다 하나의 뉴런으로 lipophilic 염료 (DiI 및 DIO)의 Microinjections. 이 분석은 라이브 배아 (손상 또는 해부)에 수행됩니다. </li></ol>

<ol>
	<li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+of+the+receptor+tyrosine+phosphatase+HmLAR2+in+a+single+cell+of+an+intact+leech+embryo+leads+to+growth-cone+collapse.">RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.</a> Current Biology. 10, 1071-1074 (2000).</li><li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Hirudo+medicinalis+DNA+Promoters+for+Targeted+Gene+Expression.">Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression.</a> Journal of Neuroscience Methods. 156, 145-153 (2006).</li><li>Gan, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+a+leech+netrin+suggests+roles+in+the+formation+of+longitudinal+nerve+tracts+and+in+regional+innervation+of+peripheral+targets.">Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.</a> Journal of Neurobiology. 40, 103-115 (1999).</li><li>Juven-Gershon, T., Cheng, S., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+super+core+promoter+that+enhances+gene+expression.">Rational design of a super core promoter that enhances gene expression.</a> Nature Methods. 3, 917-922 (2006).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Applied Physics Letters. 87, 014103 (2005).</li><li>Shefi, O., Simonnet, C., Baker, M. W., Glass, J. R., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtargeted+gene+silencing+and+ectopic+expression+in+live+embryos+using+biolistic+delivery+with+a+pneumatic+capillary+gun.">Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.</a> Journal of Neuroscience. 26, 6119-6123 (2006).</li></ol>

이 예제에서, 우리는 안으로 안으로 살고 리치 배아의 작은화된 볼륨에있는 세포에 금 입자를 제공하고 유전자를 다운 노크 공압 모세관 총을를 사용합니다. 대상 지역은 일반적으로의 직경을했다 ~ 150 μm의 및 입자가 발견되면 0과 50 μm의 사이에 조정했던 의미 침투 깊이 주변 ~ 15 μm의. 입자가 축삭지도 팩터 netrin의 dsRNA 분자로 코팅 때, 그들은 분명만을 입자를 포함하는 세포에서 발생 netrin 표현의 노크 다운 ?...

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<td>gold particles</td>
<td>Other</td>
<td>Seashell Technology, LLC</td>
<td>S1600ri</td>
<td>&nbsp;</td>
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<td>binding buffer</td>
<td>Reagent</td>
<td>Seashell Technology, LLC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>silicone rubber </td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
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localized rnai and ectopic gene expression in the medicinal leech

이 비디오에서는, 우리는 살아있는 조직에 시약의 정확한 배달을 위해 biolistic 공압 모세관 총의 사용을 보여줍니다. 우리는 내부 통제로 치료 세그먼트를 떠나, 거머리 배아들 중 선택된 세그먼트에 교란 유전자 발현 패턴에 대한 절차를 사용합니다. 공압 모세관 총을는 표본을 열지 않고 개발의 초기 단계에서 세포의 내부 레이어에 도달하는 데 사용할 수 있습니다. 살아있는 조직으로 물질화된 도입하는 방법으로, 총을 가지고 biolistic 배달 몇 가지 장점이있다 : 그것은 빠른 연락 - 무료 비파괴입니다. 또한, 하나의 모세관 총을 서로 다른 물질의 독립적인 전송에 사용될 수 있습니다. 배달 지역 ~ 50-150 μm의 크기의 측면을 가지고 있으며 0과 50 μm의 사이에서 조정하는 의미 침투 깊이, 주변 ~ 15 μm의 이상 연장하실 수 있습니다. 이 배달 세포 주사를 통해 microinjection 및 체계 최저 또는 유전 방식에 의해 허물고 하나의 세포 사이의 중간 선택한 위치에 세포의 제한된 대상 수있는 장점이 있습니다. 쓰러뜨린 또는 자연적으로 일부 중앙 뉴런에 의해 및 복부 신체 벽 표현하는 축삭지도 분자 Netrin,하지만 등의 도메인의 표현을 두드리는 소리에 대한, 우리는 그리고 신체 벽에 dsRNA 분자 또는 플라스미드 - DNA를 제공 중앙 신경. , 총을로 코팅된 입자를 로딩 (I) 특정 분석을위한 실험 설정 (가속 압력을 조정)의 준비를하고, (ii) 코팅 dsRNA 또는 DNA의 분자와 입자 (III) :이 절차는 다음 단계를 포함 을 한 분석, (IV)의 두 시약을 (입자 전달 (V) 대상 조직 (바디 벽 또는 신경)으로 코팅 입자의 제공을위한 동물을 준비하고, (VI) 배아 가공 immunostaining, immunohistochemistry와의 연결을 납품 후 2-3일 일반적으로 결과를 시각화하는) 라벨. 입자가 netrin dsRNA와 코팅 때, 그들은 분명만을 입자를 (일반적으로, 세포 당 1-2 입자)이있는 세포에서 발생 netrin 표현의 노크 다운 표시가 발생했습니다. 입자들이 핵에 정지의 연결을 세포에 플라스미드 인코딩 EGFP 유도 형광 코팅.

Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

이 비디오에서는, 우리는 새로운 초소형 유전자 총을 가진 살아있는 조직에 시약의 정확한 biolistic 전달 절차를 보여줍니다. 우리는 복부 신체 벽 및 단일 세그먼트의 신경에 dsRNA 분자를 제공함으로써 리치 태아의 축삭지도 분자 Netrin의 표현을 노크하고 있습니다.

현지화된 RNAi과 약용 거머리의 이소성 유전자 발현

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

localized-rnai-and-ectopic-gene-expression-in-the-medicinal-leech

Research

JoVE Journal

Biology

9.5K 조회수.  University of California San Diego - UCSD. 이 비디오에서는, 우리는 새로운 초소형 유전자 총을 가진 살아있는 조직에 시약의 정확한 biolistic 전달 절차를 보여줍니다. 우리는 복부 신체 벽 및 단일 세그먼트의 신경에 dsRNA 분자를 제공함으로써 리치 태아의 축삭지도 분자 Netrin의 표현을 노크하고 있습니다.

비디오: Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

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 ...

연구

생물학

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

척수 전기 생리학

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

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

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

로보틱스 및 식물 조직에서 유전자 발현의 연구 동적 이미지 분석

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

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 ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi는 Necromenic 선충류의 Microinjection에 의해 유전자 최저 및 Transgenesis을 중재 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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 ...

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.