Biz knock-ve zaman atlamalı filmleri büyüyen duyusal projeksiyonlar sağlamak için gen için yapıları üreten yardımlarından dolayı Dr. Michael Baker teşekkür etmek istiyorum. Biz Virginia Vandelinder pnömatik silah deneyleri ile teknik yardım için teşekkür etmek istiyorum.

Parçacıklar dsRNA moleküller, DNA plazmid ya da boyalar gibi farklı reaktifler, ile kaplı olabilir. Parçacıkların tipi ve çapı, reaktif ve hedef doku hücrelerinin derinliği göre seçilir: Daha büyük parçacıklar daha fazla nüfuz, ancak doku bazı hasarlara neden olabilir. 1. DsRNA kaplı partiküller hazırlanması <ol><li> Buz üzerinde% 100 izopropanol yerleştirin. </li><li> 100 ml bağlayıcı tampon; yeniden süspanse 5 mg altın parçacıkları (ortalama çapı 1.6 mm S1600ri, Seashell Technology, LLC). Çözüm hazır olduğunda, bir sonraki adıma devam edin. </li><li> Bağlayıcı tampon, vorteks kısaca ve topaklanma engellemek için 1-2 dakika sonikasyon 100 ul ekleyerek parçacık çözümü sulandırınız. </li><li> DsRNA / siRNA 5-10μg (dsRNAs / siRNA ~ 1μg/μl bir konsantrasyon için suda çözünmüş olmalıdır) ekleyin. </li><li> 2 dakika süreyle vorteksleyin ve oda sıcaklığında bekletin. SiRNA Bu konsantrasyon ve zaman aralığı sonucu altın parçacıkları doymuş. </li><li> Pelet ~ 15 sn için bir masa üstü santrifüj ~ 2500 rpm&#39;de santrifüj siRNA / altın partikül kompleksleri. </li><li> Süpernatantı ve yavaşça pelet minimal bozulma ile 750 ml soğuk izopropanol ekleyin. Kısaca Spin ve süpernatantı kaldırmak. </li><li> 100 ul soğuk% 100 izopropanol altın parçacıkları yeniden süspanse edin, sonra parçacık aglomeralar break up (2-3 bakliyat), bir banyo sonikatör kısaca sonikasyon. Son asılı parçacıkların bir cam slayt üzerine dökün ve oda sıcaklığında kurumasını bekleyin. </li></ol> 2. Belirli bir tayini için deney düzeneği hazırlama <ol><li> Her silah, en az 50μm ~ ~ 3mm arasında değişen, farklı bir meme diyafram çapı vardır. Bu nedenle silah için kullanılacak özel hedef doku alanına göre tercih edilir. </li><li> O basınç genellikle ~ 100μm (yüksek basınçlar derin parçacıklar teslim), hedef hücrelerin doku derinliği ayarlanır. Embriyo ve meme ucu arasındaki mesafe de havada altın parçacıkları gevşek bir ivme olarak, penetrasyon derinliği etkiler. </li></ol> 3. Ön kaplı parçacıkları ile silah yükleme Parçacıklar manifolduna bağlı Tygon boru parçacık enjeksiyon hatları içine bir kuru toz olarak yüklenmiş olması gerekir. Bu boru, 4 kuru bir ortamda saklanmalıdır ° C kullanımı değil, aynı reaktifleri ilave deneyler yeniden kullanılabilir zaman. Bizim deney düzeneği manifoldu ayrı bağlantı noktaları üzerinden tahlil başına iki farklı reaktifler kadar sunabilirsiniz. <ol><li> Tygon boru hattı içine yüklemeden önce, bir cam kapak slip ya da jilet kenarında kullanarak slaytlar, kurutulmuş cam kaplı parçacıkları Pençe. Bu Tygon boru çapraz kontaminasyonu engellemek için, sadece bu reaktif özel olacaktır. </li><li> Parçacıkların yük olarak, tüm boru ve konsantre yakın konektörü boyunca yayılan parçacıkları tutmak için, konektörü yakın bir U şeklinde boru viraj. Küçük bir spatula veya bir parça, katlanmış tartmak kağıt parçacıkları Pick up ve bunların yaklaşık yarısı her serisi için embriyo tüp içine yüklemek. Parçacıklar eşit olarak yaymak için yükleme adım sırasında tüp dokunun. </li></ol> 4. Hayvan Hazırlık Altın partikül teslim önce ve sonra, embriyoların oda sıcaklığında steril yapay gölet su tutulur. <ol><li> Düz bir silikon kauçuk (Sylgard 184, Dow Corning, Midland, MI), 5 mm kalınlığında bir parça oyulmuş bir oluk, embriyolar, ventral tarafına yerleştirin ve (% 8 etanol steril yapay gölet su bir çözüm onları uyuşturan aşamalarında E6-E8 genç embriyoların) deney boyunca hiçbir etanol ile yapay gölet suyu kalır. </li><li> Parçacıklar çekim hemen önce, çözümün bir parçası kaldırarak embriyoların ventral yüzeyinde ortaya çıkarmak için banyo düzeyi düşük. Stabilize etmek ve kurutma yüzeyinde tutmak için üst ortasında küçük bir delik kesilmiş bir kağıt mendil ile bir parça yerleştirin. </li></ol> 5. Hedef doku içine kaplı partikül Teslim Bir parçacıkların yük genellikle, tek bir atış genellikle birkaç yüz parçacıkların sipariş üzerine teslim ile on çekimleri için kullanılan olabilir. <ol><li> Silahı haline gelen boru hattından parçacıkların bolus enjeksiyon neden 0.3 s için vanaları açarak bir çekim oluşturun. </li><li> Çekim arasında boru duvardan parçacıklar yerinden ve böylece tabanca akışı içine enjeksiyon kolaylaştırmak için tüp hafifçe dokunun. </li><li> Ilgi doku alanında karşılamak için, birden fazla çekim gerekebilir. </li><li> Parçacık Doğumdan sonra, çok iyi bir plaka suni gölet su, tercihen de her bir embriyo embriyolar yerine geri. </li></ol> 6. İmmünoboyama ve nöronal etiketleme RNAi (ya da ektopik ifade) elde edilene kadar, embriyoların parçacık doğumdan sonra 1 ila 3 gün boyunca oda sıcaklığında ve karanlıkta saklayın. Daha sonra deney örneklerinin ve kontroller aşağıdaki yordamlardan birini yürütmek: <ol><li> Netrin mRNA varlığı ya da yokluğu assay in situ hibridizasyon. Digoxigenin etiketli sülük netrin riboprobes tüm sülük embriyolar melezleşmiştir. </li><li> Netrin protein ve nöronal doku (anti asetillenmiş tubulin) özgü diğer proteinler için tahlil İmmünositokimya. Bu prosedür tüm embriyolar da yapılır. </li><li> , Tedavi ve kontrol segmentlerinde ilgi nöronların (bizim durumumuzda mechanosensory basınç nöronlar) tam milleri etiket boya doldurur için tek bir nöron içine lipofilik boyalar (DII ve Dio) Microinjections . Bu testte, canlı embriyolarının (intakt veya disseke) yapılır. </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>

Bu gösteri, knock-canlı sülük embriyo lokalize hacmi küçük altın parçacıkları hücre içine teslim ve genler yıkmak için pnömatik kılcal silah kullandı. Hedef bölge genellikle çapı ~ 0 ile 50 mm arasında ayarlanabilir ortalama penetrasyon derinliği etrafında ~ 15 mm, 150 mm ve parçacıklar bulundu. Parçacıklar akson rehberlik faktör Netrin dsRNA molekülleri ile kaplı iken, açıkça sadece partikülleri içeren hücreler meydana gelen netrin ifade görünür knock-down neden oldu. Parçacıklar onların çekirdek...

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localized rnai and ectopic gene expression in the medicinal leech

Bu video, canlı doku içine reaktiflerin doğru Biyolistik teslimat için pnömatik kılcal silah kullanımı göstermektedir. Biz, iç kontroller gibi tedavi edilmezse segmentleri bırakarak, sülük embriyoların seçilen segmentlerde karıştırmayı gen ekspresyonu yordamı kullanın. Pnömatik kılcal silah geliştirmenin erken aşamalarında numune açmadan hücre iç katmanları ulaşmak için kullanılabilir. Maddelerin canlı dokulara lokalize giriş için bir yöntem olarak, tabanca ile Biyolistik teslimat, birçok avantajı vardır: hızlı, temas ve tahribatsız. Buna ek olarak, tek bir kılcal silah farklı maddelerin bağımsız teslimat için kullanılabilir. Teslim bölge ~ 50-150 mikron lateral boyutları var ve 0 ile 50 mm arasında ayarlanabilir ortalama penetrasyon derinliği, yaklaşık ~ 15 mikron üzerinde uzanır. Bu teslim ekstrasellüler enjeksiyonları ile mikroenjeksiyon ve sistemik demonte veya genetik yaklaşımlar yoluyla yıkmak tek hücre arasındaki ara seçilmiş bir yere hücrelerin sınırlı sayıda hedef olma avantajına sahiptir. Aşağı vurma ya da doğal olarak bazı merkez nöronları ve ventral vücut duvarı ifade edilir akson rehberlik molekül Netrin, ama dorsal etki alanı ifade çalıyor, biz ve vücut duvarı içine dsRNA veya plazmid DNA molekülleri teslim merkezi ganglionlar. Tabanca içine kaplı partiküller yükleme (i) belirli bir tayini için deney düzeneği hazırlama (hızlanan basınç ayarlama), (ii) kaplama dsRNA ya da DNA molekülleri ile parçacıklar, (iii): Bu işlem aşağıdaki adımları içerir bir tahlil, (iv) iki reaktifler (parçacık teslim, (v) hedef dokuya (vücut duvar veya ganglionlar) kaplı parçacıkların teslim hayvanların hazırlanması, ve (vi) embriyoların işleme immün, immünhistokimya ve nöronal 2 ila 3 gün sonra teslim genellikle sonuçları görselleştirmek için) etiketleme. Parçacıkları netrin dsRNA ile kaplanmış, açıkça sadece tanecikleri (genellikle 1-2 hücre başına parçacıklar) içeren hücrelerde meydana netrin ifade görünür knock-down neden oldu. Parçacıklar onların çekirdekleri durdu nöronal hücrelerin plazmid kodlama EGFP indüklenen floresan ile kaplanmıştır.

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

Bu video, biz, bir roman minyatür gen tabancası ile canlı doku içine doğru bir Biyolistik reaktifler teslim için bir yordam gösterir. Ventral vücut duvarı ve tek segmentleri ganglionlar dsRNA molekülleri sunarak sülük embriyolar akson rehberlik molekül Netrin ifade aşağı vurma.

Lokalize RNAi ve Tıbbi Sülük Ektopik Gen İfadesi

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 Görüntüleme.  University of California San Diego - UCSD. Bu video, biz, bir roman minyatür gen tabancası ile canlı doku içine doğru bir Biyolistik reaktifler teslim için bir yordam gösterir. Ventral vücut duvarı ve tek segmentleri ganglionlar dsRNA molekülleri sunarak sülük embriyolar akson rehberlik molekül Netrin ifade aşağı vurma.

Video: 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.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

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

Biyoloji

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.

Omurilik Elektrofizyoloji

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.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

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.

Bitkisel Dokular Gen İfade Araştırmalar Robotik ve Dinamik Görüntü Analizi

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.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

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.

Parafin ve Dondurulmuş Bölüm Drosophila Yetişkin Kaslar

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.

Active T. in vitro Sulandırma castaneum Telomeraz

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.

RNAi Necromenic nematodu Mikroenjeksiyon Gen demonte ve Transgenesis aracılığı ile 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.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

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.