1. Namalwa hücrelerinin hazırlanması ve sayma <ol><li> Kültür şişelerinde hücreleri çıkarın ve pelet hücreler santrifüj. PBS bilinen bir ses supernatant ve tekrar süspansiyon hücreleri çıkarın. Bu protokolde kullanılan hücreler, süspansiyon hücrelere. Yapışık hücrelere kullanıyor iseniz toplama önce trypsinize gerekecektir. </li><li> Canlı hücrelerinin sayısını belirlemek için, bir sayım slayt üzerine% 0.4 tripan mavi karışımı çözüm ve pipetlemeyin 10 ul ile 01:01 karıştırma hücre süspansiyonu bir kısım leke ve sonra TC10 otomatik hücre içine slayt eklemek sayacı (Bio-Rad Laboratories, Inc.) </li><li> TC10 hücre sayacı otofokus sayma ile devam edin, sonra en doğru saymak sağlamak ve için. Otomatik numune canlı hücreleri ve toplam hücre yoğunluğu belirler. </li><li> Viewscreen hücreleri görüntülemek için görüntüyü seçin. Sonra TC10 hücre sayacı seyreltme hesap makinesi seçeneğini seçin, otomatik olarak gerekli olacak hücre karışımı miktarını hesaplamak. </li><li> Bu deney, bir toplam üç siRNA&#39;lar kullanarak bir kontrol siRNA ve STAT6 gen, artı untransfected kontrolleri hedefleyen iki farklı siRNA&#39;lar. Bu ml&#39;de 5 x 10 6 hücre bir final konsantrasyonda 3.6 ml gerektirir. </li><li> Seyreltme hesap makinesi içine istenen konsantrasyon ve son hacim değerleri girin ve bu örnek için canlı hücre sayımı dayalı hücre süspansiyonu gerekli hacim hesaplar. Alternatif olarak, elle hesaplama yapmak. Sonra yeni bir tüp içine bu deneyler için gerekli hacim hücre karışımı aktarın. </li><li> Pelet hücreleri hücre karışımları Spin ve tüm PBS kaldırmak. Gene Verici elektroporasyon tampon (Bio-Rad Laboratories) 3.6 ml süspanse edin. 800 ul alikotları uygun siRNA&#39;lar içeren tüp içine aktarın ve hafifçe karıştırın. Hücreler artık elektroporasyon için hazırız. </li></ol> 2. 96-iyi Tabaklar hücreleri Electroporating <ol><li> 96-iyi elektroporasyon plaka birlikte electroporated dört tedaviler için çoğaltır sağlar. </li><li> 96-iyi elektroporasyon plaka uygun kuyu içine hücre süspansiyonu 150 ul aktarın. </li><li> MXcell elektroporasyon plaka odasında plaka yerleştirin ve kapağını kapatın. </li><li> Kullanılan hücre hattı ve tampon için optimize edilmiş ayarlar kullanarak Electroporate. Bu hücreler, bir üstel bozulma dalga, 250 V cihaz ayarları ile 350 μF, ve 1000 Ω direnç kullanarak electroporated. </li><li> Elektroporasyon tamamlandıktan sonra, her bir kuyu içinde karıştırın ve daha sonra önceden ısıtılmış medya içeren bir doku kültürü plaka hücreleri aktarmak için hafifçe aşağı yukarı pipet ve. Namalwa hücreleri RPMI (Life Technologies)% 7.5 ve% 1 FCS piruvat ile desteklenmiş yetiştirilmektedir. </li><li> Electroporated henüz kontrol numuneleri alınmış ve kalan hücre süspansiyonu electroporated hücrelere aynı kültürü, önceden ısıtılmış kültür orta yemekleri eklenir. </li><li> 37 geceleme inkübe hücreleri ° C ile% 5 CO 2. </li><li> 24 saat sonra hücrelerin bir set 10 ng / ml son konsantrasyon rekombinant IL-4 (R &amp; D Systems) ile sağlandı. Kontrol hücrelerinin ikinci sette sadece ek ortam alırsınız. Tüm hücreler 37 ° C ve% 5 CO 2 ek bir 24 saat inkübe edilir. </li></ol> 3. RNA ayıklayın ve kontrol <ol><li> Büyüme olmak üzere toplam 48 saat sonra, her bir kuyunun hücrelerin sayısı, ve her biri de bir kısım transfer RNA ekstraksiyonu için bir mikrofuge&#39;de tüp ve batı blot veya diğer protein analizi için ikinci bir kısım dondurmak. </li><li> Pelet hücreleri örnekleri santrifüj, ve sonra her bir tüpten süpernatantı çıkarın ve atın. </li><li> RNA ekstraksiyonu ile devam edin. Biz genellikle dahil protokole göre Aurum toplam RNA kiti (Bio-Rad) kullanabilirsiniz. </li><li> SiRNA aracılı gen demonte izlerken bozulmuş RNA yanıltıcı sonuçlar verecektir, çünkü bu RNA kalitesini doğrulamak için özellikle önemlidir. 260/280 oranı RNA bütünlüğü anlamına gelmez, bu nedenle RNA bozulmuş değil onaylamak için bir jel ya da mikroakışkan cihaz örnekleri çalıştırmak için önemlidir. </li><li> Tek adımda hem kalite ve miktar değerlendirmek için, bu örneklerin Experion otomatik elektroforez sistemi (Bio-Rad) kullanılarak analiz edilecektir. </li><li> İlk olarak, ısı örnekleri. Sonra, başbakan, yük ve vorteks Experion RNA çip. Experion elektroforez istasyonu içine çip takın ve çalıştırın. </li><li> Çalıştırmak tamamlandıktan sonra, Experion yazılım (geleneksel bir RNA benzer bir Elektroferogram, sonuçlar tablo şeklinde (RNA konsantrasyon, ribozomal oranı ve RNA kalite göstergesi numarasını görüntüler) ve sanal jel sonuçları otomatik olarak gösterecektir jel). Yüksek kalitede toplam RNA örnekleri genellikle 8-10 RQI olacaktır. </li&gt; </ol> 4. Real-time PCR <ol><li> RNA kalitesi doğruladıktan sonra, cDNA reaksiyonlar ile devam edin. Bu deney bir tepki olarak cDNA sentezi ve real-time PCR iScript bir adım RT-PCR karışımı (Bio-Rad) birleştirmek için kullanılır. </li><li> Her astar kümesi için RNA şablonlar dışında tüm reaksiyon bileşenlerini içeren bir master miks olun ve PCR plakasına bu dağıtmak. </li><li> RNA yeknesak bir konsantrasyona seyreltilmesi sonra, uygun kuyulara RNA örnekleri ekleyin. Her reaksiyon üç nüsha olarak kurulur. </li><li> Plaka Seal, ve CFX384 gerçek zamanlı PCR sistemi (Bio-Rad) içine yerleştirin. Uygun protokolü seçin ve çalışma başlar. </li><li> Çalıştırmak tamamlandıktan sonra, gen ekspresyonu kontrol siRNA electroporated hücreler aynı normalleşme deneysel siRNA electroporated hücreleri bir referans gen normalize ilgi gen gen ekspresyonu karşılaştırılarak belirlenir. </li></ol> 5. SiRNA demonte Analizi <ol><li> CCL17 ifadesi CCL17 IL-4 bağımlı indüksiyon transkripsiyonel aktivatör STAT6 rolünü araştırmak için kantitatif gerçek zamanlı PCR kullanarak takip edildi. Bu deneme için, GAPDH bir referans gen olarak kullanılmıştır. Alternatif referans gen ya da birden fazla referans genler aynı şekilde kullanılabilir. </li><li> CFX Manager yazılımı otomatik olarak aynı örnek referans gen ekspresyonu CCL17 ifade normalize ve basitçe, &quot;referans&quot; ve kontrol tedavisi (IL-4 indüksiyon olmadan kontrolü siRNA) olarak GAPDH seçerek tedavileri kontrol etmek için otomatik olarak normalize değerleri karşılaştırıldığında CFX yöneticisi yazılımı deney ayarları penceresi &quot;kontrol&quot;. </li><li> Olmadan, IL-4 indüksiyon, CCL17 transkripsiyonunu kontrol tedavisi ile karşılaştırılabilir, bünye düşük seviyelerde kaldı. </li><li> IL-4 ile indüklenen ve normal bir STAT6 yolağı (yani, kontrol siRNA veya untransfected kontrol tedavileri) istinat Hücreler CCL17 ifade 8-10 kat indüksiyon gösterdi. </li><li> IL-4 ile ancak STAT6 ifade ya STAT6 siRNA tanıtımı tarafından inhibe uyarılan hücreler CCL17 IL-4 kaynaklı ifade STAT6 rolünü teyit uninduced hücreleri sadece yaklaşık 2 kat daha yüksek CCL17 ifade gösterdi. </li></ol> 6. Temsilcisi Sonuçlar <img src="/files/ftp_upload/1904/1904fig1.jpg" alt="Şekil 1" /> Şekil 1. IL-4 sinyal yolağı Genel Bakış. IL-4 - kendi reseptörüne A) Cilt STAT6 dimerization ve aktivasyonuna yol açar. STAT6 translocates çekirdeği etkinleştirilir ve bu tür CC17 olarak hedef genlerin transkripsiyonunu aktive eder. B) IL-4 indüksiyon olmadan, CCL17 transkripsiyonunu, bünye düşük seviyelerde kalmaya devam edecektir. C) siRNA-aracılı STAT6 gen susturulması sonra, IL4 tedavisi çok az veya hiç artış CCL17 ifade yol açacaktır. <img src="/files/ftp_upload/1904/1904fig2.jpg" alt="Şekil 2" /> Şekil 2. IL4 tarafından indüksiyonu CCL17 kantitatif gerçek zamanlı PCR ile farklı koşullar ile ölçülür. A) IL-4 olmadan yetişen Örnekleri gen ekspresyonu ne olursa olsun, düşük bir ifade kurucu seviyesi vardı. B) yeşil ve mavi de görüldüğü gibi, kontrol örnekleri IL-4 ile tedavi normal yanıt verdi; CCL17 8-10 kat indüksiyon ile. Ancak, hücreler, STAT6, kırmızı ve pembe gösterilen hedef siRNA&#39;lar ile transfekte fikri destekleyen, IL-4 yanıt CCL17 ifade azalmıştır STAT6 CCL17 IL-4 kaynaklı ifade bir rol oynar.

Yazar, bu makalede kullanılan reaktifler ve aletleri üreten Bio-Rad Laboratories tarafından istihdam edilmektedir.

Bu makale, süspansiyon hücre hattı transfect ve gen ekspresyonu bu hücrelerin nasıl takip etmek zor bir içine nasıl siRNA&#39;lar electroporate göstermiştir. Bu işlemi yaparken tüm örnekleri arasında mümkün olduğunca tutarlı bir şeyleri saklamak için hatırlamak önemlidir.

using an automated cell counter to simplify gene expression studies: sirna knockdown of il-4 dependent gene expression in namalwa cells

SiRNA aracılı gen demonte gen ekspresyonu çalışmalarında önemli bir araç olmaya devam etmektedir. sadece tek genlerin downregulating etkilerini incelemek için siRNA çalışmalar yürütülmektedir değil, aynı zamanda sinyal yollar ve diğer karmaşık bir etkileşim ağları sorgulamak için. Bu yolağı analizleri, ilgili cep telefonu modelleri kullanımı ve hücresel fizyoloji az pertürbasyon neden yöntemleri de gerektirir. Elektroporasyon giderek soruşturma altında sinyal yolağı değiştirmeden hücre hatları ve birincil hücreler transfect zor siRNA ve diğer nükleik asitlerin tanıtmak için etkili bir yol olarak kullanılmaktadır. Başarılı bir siRNA deneyi için birden fazla kritik adımlar vardır, ve çeşitli deneysel aşamada veri kalitesini arttırırken, çalışmalarını kolaylaştırmak için yollar vardır. SiRNA aracılı gen demonte proje ile başlamanıza yardımcı olmak için, biz yazılan transfeksiyon real-time PCR gen ekspresyon analizi yoluyla elektroporasyon önce hücrelerin toplanması ve sayma komple bir yol çalışması gerçekleştirmek için nasıl göstereceğiz. Aşağıdaki çalışmada, IL-4 Burkitt lenfoma hücre hattı (Namalwa) CCL17 bağımlı gen ekspresyonu transkripsiyonel aktivatör STAT6 rolünü araştırıyor. SiRNA tabanlı deneyler yapışmış ve süspansiyon hücreleri hem de geniş bir yelpazede ortaya teknikler yararlıdır. Ayrıca MXcell elektroporasyon sistemi aynı anda kullanarak birden fazla numune nasıl electroporate TC10 otomatik hücre sayıcı sayma hücre, RNA Experion otomatik elektroforez sistemi ile kalite ve miktar nasıl aynı anda değerlendirmek için nasıl düzene gösterecektir.

Watch this Scientific Journal Video about Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells at JoVE.com

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

Bu prosedür, hücre hatları transfect ve gerçek zamanlı PCR ile gen ekspresyonu takip etmek zor içine siRNA tanıtmak için hızlı ve kolay bir iş akışını açıklar. Otomatik bir hücre sayıcı kullanımı, çok iyi elektroporasyon plaka ve otomatik elektroforez istasyonu pahalı robot işleme gerek kalmadan hızlı ve güvenilir sonuçlar sağlar.

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 ...

using-an-automated-cell-counter-to-simplify-gene-expression-studies

Universidad San Sebastian

Research

JoVE Journal

Biology

15.5K Görüntüleme.  Bio-Rad Laboratories.  中文 

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

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Fare Makrofagların yılında Doğuştan Bağışıklık Tepkisi Soruşturma RNA girişimi kullanma

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

İmmünoloji ve Enfeksiyon

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Nucleofection İnsan THP-1 Makrofagların Yüksek Verimli Transfeksiyonu

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Antisense Oligonucleotides as a Tool for Prolonged Knockdown of Nuclear lncRNAs in Human Cell Lines

Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a substantial fraction of lncRNA preferentially accumulates in the nucleus. For analysis of the function of nuclear lncRNAs, it is important to achieve efficient knockdown of these transcripts inside the nucleus. In contrast to the use of RNA interference, a technology that depends on the cytoplasmic silencing machinery, an antisense oligonucleotide (ASO) technology can achieve RNA knockdown by recruiting RNase H to the RNA-DNA duplexes for nuclear RNA cleavage. Unlike the use of CRISPR-Cas tools for genome engineering, where possible alterations in the chromatin state can occur, ASOs allow the efficient knockdown of nuclear transcripts without modifying the genome. Nevertheless, one of the major obstacles to ASO-mediated knockdown is its transitory effect. For the study of long-lasting effects of lncRNA silencing, maintaining efficient knockdown for a long time is needed. In this study, a protocol was developed to achieve a knockdown effect for over 21 days. The purpose was to evaluate the cis-regulatory effects of lncRNA knockdown on the adjacent coding gene RFC4, which is related to chromosomal instability, a condition that is observed only through time and cell aging. Two different human cell lines were used: PrEC, normal primary prostate epithelial cells, and HCT116, an epithelial cell line isolated from colorectal carcinoma, achieving successful knockdown in the assayed cell lines.

To analyze the function of lncRNAs in time-dependent processes such as chromosomal instability, a prolonged knockdown effect must be achieved. To that purpose, presented here is a protocol that uses modified antisense oligonucleotides to achieve effective knockdown in cell lines for 21 days.

Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a ...

Kanser Araştırmaları

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

Mikroakışkan Cihaz Kullanılarak siRNA Yüklü Lipid Nanopartiküllerinin Üretimi

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

Biyokimya

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 ...

Biyoloji

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Canlı Hücre Kalsiyum Görüntüleme siRNA aracılığı ile gen susturma ile birlikte Ca tanımlar 2 + ER Membran Kaçak Kanallar ve Düzenleme Mekanizmaları

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Fare Embriyonik Kök Hücre Bakım ve Kendini yenileme Regulate Genler Okumak Oct4GiP Reporter Assay

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4 Bağlı Hücre Ölümü üzerine Nakavt Etkilerinin Gen Ekspresyonu ve Ölçüm Şartlı Nakavt için Hücre hatları hazırlanması

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

Yüksek içerik, Open Source Software tarafından desteklenen Evrensel Mikroskop Verilerden Floresan Göstergeler, Bireysel Hücre Niceleme için iş akışı

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3&#39;UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into &#62;75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Adenofection: tükenmesi-Kurtarma Experiments Hücresel Morphodynamics Moleküler şaperonlar Rolü incelenmesi için Bir Yöntem

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

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

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