Bu araştırma çabaları Kimya, Biyomühendislik, Çevre ve ödül numaraları CBET-0853780 ve CBET-1159344 ve Ulusal Sağlık Enstitüleri, Çevre Sağlığı Bilimleri Ulusal Enstitüsü (NIEHS) altında Transport (CBET) Sistemleri, Ulusal Bilim Vakfı Bölümü tarafından desteklenmiştir ödül sayısı 1R43ES022567-01 ve Ulusal Kanser Enstitüsü, ödül sayısı CA127745-01 altında Kanser Görüntüleme Programı kapsamında. Bu çalışmada kullanılan IVIS Lumina aracı ABD Ordusu Savunma Üniversitesi Araştırma Araçları Program elde edilmiştir.

1. Hücre Hazırlanması <ol><li> Sıvı azot donmuş stok biyolüminesans HEK293 hücreleri bir şişe elde etmek ve Dulbecco&#39;nun Modifiye Edilmiş Eagle% 10 cenin sığır serumu ile desteklenmiş ortamı (DMEM), 0.01 mM esansiyel olmayan amino asitler, 1 x antibiyotik antimikotik ve T, 0.01 mM sodyum piruvat onları büyümeye 37, 75 doku kültürü şişesi ° C ve% 5 CO2. Not: Medya ve ek parçaları hücre hatları göre değişir ve bu nedenle buna göre seçilmelidir. </li><li> Yenile orta her kapsamlı kadar 2-3 gün ~ 80% izdiham. Not: gerekli hücrelerin miktarı deneysel tasarım dayalı olacaktır. Gerekirse, daha fazla hücre alt kültüre ile elde edilebilir. </li><li> Testleri için hasat hücreleri. <ol><li> Orta kaldırmak ve fosfat hücreleri yıkayın şişeye yavaşça dönen ve daha sonra çıkarılır ve PBS ile peynir salin (PBS). </li><li> 2 dakika boyunca 37 ° C&#39;de inkübe tripsin ve eklemeveya hücre kadar şişesi müstakil. </li><li> PBS içinde müstakil hücreleri toplamak ve temiz bir santrifüj tüpü içine aktarabilirsiniz. Not: nonadherent hücre hatları santrifüj, ayrı hücrelere kullanılan ve PBS ile bir kez yıkayın varsa. </li></ol></li><li> Hemasitometre ya da başka hücre sayımı sistemi kullanılarak toplam hücre sayısı belirlenir. </li><li> 5 dakika boyunca 300 x g&#39;de hücre süspansiyonu santrifüj. Süpernatantı atın ve taze, önceden ısıtılmış orta hücreleri tekrar süspansiyon. </li><li> Opak bir çok-çukurlu plaka bireysel kuyular içine hücre çekirdeği eşit sayıda. Bu örnekte, plaka 5 x 10 siyah 24 oyuklu plakanın her kuyuya, 1 ml&#39;lik bir hacimde 5 hücreleri. Plaka üç kopya halinde kuyularda ortamına ait eşit bir hacmi negatif kontrol olarak hareket eder. Not: her iyi kaplama hücrelerin sayısı esnek ve ayrı deneyler için optimize edilebilir. Her bioluminescent hücre hattı için, deneysel hücre sayısı ve biolumi arasındaki ilişkiyi belirlemeknescent çıkış yanı sıra, en az saptanabilir hücre önce herhangi bir toksisite testleri sayılır. Standart koşullar altında görüntüleme nüfusun boyutlarda geniş bir aralığı (örneğin, 1 x Ekim 3-1 x 10 hücre 6) karşısında yer alan bu tedbir, biyolüminesans yapmak için. </li><li> Aşağıda tarif edildiği gibi test bileşik (ler) ile birlikte hücre tedavisi. </li></ol> 2. Kimyasal Hazırlama <ol><li> Bir konsantre stok çözeltisi olarak test edilen kimyasal hazırlayın. Bu örnekte, bir 100 mg / bleomisin ailesinin bir antibiyotik ml stok çözeltisi kullanın. Not: organik bir çözücü kimyasal eklenmesi için bir araç olarak kullanıldığında, özellikle de potansiyel taşıyıcı bazlı toksik etkilerini minimuma indirmek için, stok konsantrasyonu, en az 1000 kat, istenen uygulama sonrası konsantrasyon olması önerilir. </li><li> Doğrudan hücrelere hazırlanan kimyasal stok ekleyin. Bu örnekte, doz nihai konsantrasyonları 100, 200, 300, ve tri 400 ug / ml &#39;de ilgi antibiyotikkuyu plicate. Tedavi edilmeyen kontrol olarak hücreler üç kuyu bırakın. Not: Hedef kimyasal nihai konsantrasyon spesifik deneysel hedeflerine göre seçilmelidir. Konsantrasyonları geniş bir aralık, normalde toksik etkilerin tam bir spektrum elde etmek için test edilir. </li></ol> 3. Görüntüleme ve Veri Analizi <ol><li> Hemen kimyasal ilave edildikten sonra, görüntü elde etme ve biyolüminesans ölçümü için uygun enstrümanın görüntüleme odasında plaka yerleştirin. </li><li> Her okuma için bir 10 dakika entegrasyon zaman kullanarak 24 saatlik süre içinde önlem biyolüminesans her 15 dk. Not: Bu örnekte kullanılan entegrasyon zaman bir başına plaka bazında ve seçtiğiniz diğer luminometreyle kullanılarak her iyi bazında ölçümü için ayarlanabilir. Entegrasyon süresi seçilen bioluminescent hücre hattı dayalı da ayarlanabilir. Biyolüminesans hücre yıkımı ya da herhangi bir dış uyarı olmaksızın özerk üretilir çünkü okumakbulgular spesifik deney amaçları yerine getirmek için arzu edilen herhangi bir zaman noktasında alınabilir. </li><li> Uyumlu yazılım kullanarak ilgi bir bölge belirleyerek her kuyudan bioluminescent yoğunluğunu ölçmek. Ya toplam akı (fotonlar / saniye) veya her numunenin (fotonlar / cm / saniye 2 / steridian) olarak ışık çıkışı görüntüleyin. </li></ol>

<ol>
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M., 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=Autonomous+bioluminescent+expression+of+the+bacterial+luciferase+gene+cassette+(lux)+in+a+mammalian+cell+line.">Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.</a> PLoS ONE. 5, e12441 (2010).</li><li>Close, D. M., Hahn, R. E., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+toxicant+bioavailability+using+a+constitutively+bioluminescent+human+cell+line.">Determining toxicant bioavailability using a constitutively bioluminescent human cell line.</a> , 1-4 (2011).</li><li>Oliva-Trastoy, M., Defais, M., Larminat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+the+antibiotic+Zeocin+by+stable+expression+of+the+Sh+ble+gene+does+not+fully+suppress+Zeocin-induced+DNA+cleavage+in+human+cells.">Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.</a> Mutagenesis. 20, 111-114 (2005).</li><li>Close, D. M., Webb, J. D., Ripp, S. A., Patterson, S. S., Sayler, G. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+optimized+bacterial+luciferase,+firefly+luciferase,+and+green+fluorescent+protein+for+continuous+imaging+of+cell+culture+and+animal+models.">Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models.</a> J. Biomed. Opt. 16, e12441 (2011).</li></ol>

DM Kapat, SA Ripp ve GS Sayler 490 BioTech, Inc kurucuları ve sahipleri

Bu yöntem, canlı hücreler sürekli olarak kullanım süresi boyunca takip edilmesine olanak in vitro sitotoksisite tarama deneyi, bir özerk olarak biyolüminesans memeli hücrelerinin kullanımını gösterir. Bu protokol, esnek ve gerektiğinde gibi belirli deney koşulları karşılamak için modifiye edilebilir. Örneğin, deney akut toksik etkilerini izleme için uygundur Burada yer ama art arda artan zaman aralıklarında (yani her 24 saat) görüntüleme ve arasında standart enküba...

Onlar Gıda ve İlaç İdaresi tarafından tüketici kullanım için onaylanmıştır önce ABD&#39;de, insan tüketimine yönelik ilaç ve diğer ürünleri kapsamlı bir değerlendirme gerektirir. Bu testinin yapılması için mali yük büyük ölçüde tüketiciler için artan maliyet yeni bileşik geliştirme ve bu nedenle çevirir maliyetini artırır geliştirici 1, yerleştirilir. Bu tarama geleneksel olarak çok insan ana için temsilci olarak vekaleten Hayvanlar üzerinde kullanılan olsa da, bu (Tox / ADME adsorpsiyon, dağılım, metabolizma, boşaltım ve toksisite her yıl harcanan yaklaşık 2800000000 $ ile, büyük bir mali yük olduğu kanıtlanmıştır ) Tek başına tarama 2 ve hayvan modellerinde güvenilir insan toksikolojik yanıtları 3 tahmin edemez düşündüren montaj kanıtlar. Bu nedenle, in vitro insan hücre kültürü tabanlı test nedeniyle nispeten daha düşük olan son yirmi yılda popülerlik kazanmıştırmaliyet, daha yüksek verim ve insan biyoyararlanım ve toksikoloji 4 daha iyi temsil. Mevcut hücre kültürü bazlı toksisite tarama yöntemleri hücre canlılığı 5 değerlendirmek için, hücresel membran bütünlüğünü sondalama veya mitokondriyal aktivite düzeyi izleme, endojen olarak mevcut sitoplazmik enzimlerin aktivitesinin taranması, örneğin ATP seviyelerinin ölçümü gibi, uç noktalar, istihdam , 6. Ölçümler ve böylece tek bir zaman noktasında veri üreten alınabilir önce Ancak, ne olursa olsun seçilen nokta dolayı, bu yöntemler her numunenin imha gerektirir. Sonuç olarak, çok sayıda numune hazırlanmış ve temel toksikolojik kinetik çalışmalar için paralel olarak tedavi, daha yeni bir bileşik geliştirilmesi için gerekli maliyet ve iş gücü ekleyerek olması gerekir. Alternatif olarak, bu tür tahliller kullanılarak Gaussia 7 lusiferaz, Vargula lusiferaz 8 ve 9 Metridia lusiferaz gibi lusiferaz salgılananhücre parçalama için olan ihtiyacı ortadan kaldırır ve son nokta ölçümü için, ortamın bir kısmını gerektirir, ancak, yine de bu önceden belirlenmiş zaman noktalarında, örnekleme ile sınırlıdır ve ayrıca dışsal ışık aktive edici yüzeyler ilavesi gerektiren geliştirilmiştir. Örnek gerekli imha zararına yanı sıra substratlar maliyeti ortadan kaldırmak için önlemek için, bir insan hücre hattı benzer canlı hücreler sürekli olarak izlenmesi için izin vermek için tam bakteriyel biyolüminesans (lux) gen kasetinin (luxCDABEfrp) ifade eden tasarlandı floresan-boya tabanlı canlı hücre görüntüleme için, ancak ek foton-aktive edici ve mikroskobik inceleme prosedürler olmaksızın. Bu hücre hattı, böylece numunenin imha kaçınarak, yapısal olarak dış uyarılması için gerek olmaksızın sürekli, doğrudan saptanması için bir optik sinyal üretme yeteneğine sahiptir. Mekanik olarak, bu hücrelerden üretilen bioluminescent sinyal nedens luxAB şekillendirilmiş lusiferaz enzim indirgenmiş riboflavin fosfat varlığında (FRP geni tarafından FMN ikinci geri kazanılır FMNH 2, uzun zincirli bir yağ aldehid (endojen maddeleri kullanarak luxCDE gen ürünleri tarafından sentezlenen ve rejenere) oksidasyonunu katalize zaman ürün) ve moleküler oksijen 10. Konakçı hücre içinde, lux kaset ekspresyonu bu nedenle üretilen ve hücre yıkımı veya eksogen bir substrat ilave edilmeden tespit edilmesi ışık sağlar. Benzer şekilde, lux genler ve endojen olarak mevcut FMN ve O 2 cosubstrates ve FMNH 2 FMN dönüşüm destekleyen bir ortamın bakım gereksinimi, arasındaki etkileşim, elde edilen biyolüminesans sinyali sadece oturma tespit edilebilir olmasını sağlar , metabolik olarak aktif hücrelerin. Bu gereksinimler daha önce o lüks-bas göstermek için istismar edilmiştired bioluminescent çıktı hücresel nüfus büyüklüğü 11 ve zehirli bileşik maruz kalma bir doz-yanıt moda 12 autobioluminescent üretim bozar ile güçlü ilişkilidir. Burada toksisite testi için autobioluminescent memeli hücrelerinin uygulama doğrulamak için temsili bir örnek olarak bilinen DNA hasarına aktivitesi ile bleomisin ailesinin bir antibiyotik otomatik toksikolojik tarama göstermek için, daha önce karakterize edilen autobioluminescent insan embriyonik böbrek (HEK293) hücre hattı 11 kullanır.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

Memeli hücre tabanlı in vitro deneyleri yaygın olarak toksikolojik çalışmalar için hayvanlar üzerinde test alternatif olarak istihdam edilmiştir ama nedeniyle ateşböceği lusiferaz-tabanlı tarama yöntemlerinin yıkıcı doğası zorunlu paralel numune hazırlama, yüksek para ve zaman maliyetleri nedeniyle sınırlı kalmıştır . Bu video ilgi çekici bir bileşik olan sitotoksik etkilerinin izlenmesi için bir ucuz ve kolay bir yöntem olarak, bir lusiferin substratının yıkıcı ilave gerektirmeyen Bağımsız biyolüminesans memeli hücrelerinin kullanımını tarif etmektedir. Stabil bir şekilde tam bakteriyel biyolüminesans (luxCDABEfrp) geni ifade kaseti, memeli hücreleri bağımsız olarak bir optik sinyal üreten bir harici enerji kaynağı, ya da geleneksel bir numunenin tahrip edilmesi pahalı ve muhtemelen müdahale lusiferin substratının, uyarma ilavesi olmadan 490 nm pik optik görüntüleme işlemi sırasında yapılans. Dış stimülasyon gelen bu bağımsızlık çıkan sinyal sadece aktif metabolizma sırasında tespit, yani sadece hücre bioluminescent reaksiyon korumak için yük getirmektedir. Biyolüminesans üretiminde değişimler hücre büyümesi ve metabolizması üzerindeki olumsuz etkilerin bir göstergesi olduğundan, bu özellik, lux-eksprese eden hücre soyu sitotoksik etkilerine karşı biosentinel olarak kullanım için mükemmel bir aday yapar. Benzer bir şekilde, gerekli örnek imha özerk doğası ve eksikliği zehiri maruz kalma süresi boyunca gerçek zamanlı olarak aynı numune üzerinde tekrar görüntüleme izin verir ve otomatik bir şekilde mevcut görüntüleme ekipmanı kullanarak çoklu örnekler yardımıyla gerçekleştirilebilir.

Bu çalışmada, autobioluminescent HEK293 hücreleri ve dinamikleri spektrumlu antibiyotik (Şekil 1) tepki olarak 24 saatlik bir süre boyunca sürekli olarak izlenmiştir. Bağlanabilen ve DNA 13 yarılması ile, canlı hücreleri öldürmek için bilinen bleomisinin ailesinin bir üyesi olan bu antibiyotik, toksik etkileri, Pseudocolor görüntü doğrudan görselleştirilebilir tedavi edilmemiş hücrelere kıyasla, biyolüminesans üretiminde bir azalma ile ortaya konmuştur (Ş...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Bakteriyel biyolüminesans gen kasetinin ifade eden memeli hücreleri (<em&gt; Lux</em&gt;) Özerk ışık üretirler. Kimyasal maruz kalma üzerine çıkan biyolüminesens dinamikleri bu hücrelerin kolayca yüksek verimli otomasyon için adapte edilebilir bir ucuz, sürekli, gerçek zamanlı toksisite tarama aracı yapma, hücresel büyüme ve metabolizma üzerindeki tedavi etkilerini yansıtacak şekilde gösterilmiştir.

Sitotoksisite Sürekli ve gerçek zamanlı izlenmesi için özerk Bioluminescent Memeli Hücre

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

autonomously-bioluminescent-mammalian-cells-for-continuous-real-time

Research

JoVE Journal

Biology

10.0K Görüntüleme.  Oak Ridge National Laboratory. Bakteriyel biyolüminesans gen kasetinin ifade eden memeli hücreleri ( Lux) Özerk ışık üretirler. Kimyasal maruz kalma üzerine çıkan biyolüminesens dinamikleri bu hücrelerin kolayca yüksek verimli otomasyon için adapte edilebilir bir ucuz, sürekli, gerçek zamanlı toksisite tarama aracı yapma, hücresel büyüme ve metabolizma üzerindeki tedavi etkilerini yansıtacak şekilde gösterilmiştir.

Video: Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Memeli Hücreleri Trypsinizing ve alt kültüre

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Biyoloji

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

Memeli Hücreleri RNAi Tarama MİSYON esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Kantitatif Gerçek-zamanlı PCR (qPCR) Diziler kullanarak Pre-mikro RNA&#39;lar ve microRNA, Profilleme

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

Floresans Rezonans Enerji Transferi Ligand-reseptör etkileşimleri gerçek zamanlı izleme

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

Unimoleküler Biyosensörler kullanma Canlı Hücreler Olaylar Sinyal gerçek zamanlı izlenmesi için Mikroskopi FRET

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Floresan Mikroskobu ile Çoklu Nesiller boyunca Canlı Memeli Hücrelerinde Plazmid Çoğaltma İzlenmesi

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-tabanlı Real-time PCR microRNA Kantitatif Ölçüm Yaklaşımları

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Bakteri, Maya ve İnsan Hücrelerinde Geçici Olarak Oluşan Moleküler Şaperon Meclislerinin Yerinde İzlenmesinde

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...