Yazarlar Dr teşekkür etmek istiyorum. Başlangıçta floresan mikroskopi LAS eğitim için Philina S. Lee ve Alan D. Grossman. Yazarlar ayrıca teşekkür Dr. Yardım ve ipuçları görüntüleme için Melanie Berkmen ve Hajime Kobayashi. Bu çalışma, Edebiyat, Fen-Edebiyat Fakültesi ve Michigan Üniversitesi, Moleküler, Hücresel ve Gelişimsel Biyoloji Bölümü start-up fonları tarafından desteklenen oldu.

Mikroskopi için hücrelerin Büyüyen kültürlerin 1. Bir veya iki gün önce görüntüleme, B. hazırlamak subtilis görselleştirmek için istediğiniz translasyonel füzyon proteini içeren süzün. Adımları 1A ve 1B Deneme turlarında, büyüme durumu suşu en iyi görüntüyü sağlayan belirlemek için gerekli olacaktır. Çoğu durumda, hücreler, üstel büyüme aşamasında görüntülü olmalıdır. <ol class="ualpha"><li> Bazı B. subtilis suşları (endojen lokustaki ilgi ve GFP geninin arasında translasyonel füzyon entegrasyonu nedeniyle yetersiz büyümeye, özellikle suşları), görüntüleme için iki gün önce ilk büyüme, yüksek kaliteli görüntüler için gerekli . Ilk gününde, çizgi B. subtilis seçici antibiyotik (ler) ile LB agar yansıması zorlanma ve 30 ° C&#39;de bir gece inkübe Ikinci gününde, tek bir koloni ile 100 ul 0.85% serum fizyolojik aşılamak ve 30 gece inkübasyon tarafından her seyreltme 100 mcL kaplama LB agar plakaları üç 10 kat seri dilüsyonları (seçici antibiyotikler ile), ° C gerçekleştirmek Üçüncü gününde, hücrelerin ışık konfluent büyüme ile seyreltme plaka seçin. Bir tabak Işık konfluent büyüme potansiyel (adım 4) mükemmel görüntüleme sonuçlar, katlanarak büyüyen sağlıklı bir başlangıç ​​inokulum ile büyüme ortamı sağlayacaktır. Bu adım, leke FM4-64 ile en yüksek kaliteli membran görüntüleme için yararlıdır. </li><li> B. Diğer subtilis suşları sadece steril bir çubuk görüntüleme için önceki gün ile tek koloniler için seçici LB agar uygulanacak ve 30 ° C&#39;de bir gece inkübe Ertesi gün, bu plaka üzerinde hücreler (adım 4) görüntüleme için bir inokulum olarak kullanmak yeterli olacaktır. NOT: B. subtilis, sıvı bir ortamda geceleme kültürleri 1 lag fazı büyüme bir süre neden spor oluşumu ve yeter tepkiler, bir kombinasyonu nedeniyle uygun değildir. </li></ol> 2. Görüntüleme, yerine 10 ml, 125 ml steril Erlenmeyer şişesinin içine S7 50 1-3 orta sabahı . 3. Inokulum elde etmek için tek koloniler veya ışık konfluent hücreleri LB agar plaka S7 50 orta 2 ml kadar hücrelerin yeniden süspanse edin. 4. ~ 0.08 0.1 ila 600 nm dalga boyunda ölçülen bir ilk optik yoğunluk (OD 600) ile 10 ml S7 50 orta bir kültür içeren Erlenmeyer şişesi yeterli S7 50 orta hücre inokulum ekleyin. Bu aşamada, en az 10 kat hava orta oranı olması önemlidir. Böylece, 10 - 12.5 ml 125 ml Erlenmeyer şişesinin inoküle orta kullanın. 5. Kültür OD 600 ~ ulaşır 0.5-0.8 ° C ye kadar her 30 kültür büyütün. Çalkalamalı su banyosu, 150-200 dakikada devrimlerle tercih edilmektedir. DNA&#39;nın zarar veya uyumsuzluğu indükleyici ajanlar ile meydan Kültürler erken bir üstel büyüme aşamasında olmalıdır OD 600 hücrelerin (bkz. Tablo 1), tedavi için gerekli olan büyüme ek süre ile OD 600 hedefe ulaşmak olacağını , Örneğin, 2-aminopurine bir tedavi saat, 2 aminopurine ile tedavi edilen bir kültür ve bu nedenle tedavi bir OD 600 / büyüme sonuçlar bir saat ~ 0.5-0.8 gibi, OD 600 ~ 0.3-0.4 olmalıdır gerektirir. sayede hücreleri görüntüleme için hazır. Numune Hazırlama 1. Pipet, 1.5 mL mikrosantrifüj tüpe kültür hücreleri 300 ul. 2. Hücre zarının görüntüleme isteniyorsa, 1 mg / ml stok solüsyonu leke FM4-64 membran (Invitrogen) her numune için 1:1000 dilüsyon ekleyin. FM4-64 A titrasyon istenen floresan sinyal elde etmek için gerekli olabilir. Tipik bir titrasyon aralığı, 1:100, 1:1000 ve 1:10,000. 3. Örnekleri, aşağıda açıklandığı gibi slayt 1X Spizizens orta% 1 agaroz ile hazırlanmakta olan ise, on dakika kadar veya oda sıcaklığında oturmak (bkz: adım 1 &quot;slayt hazırlama&quot;) izin verin. 4. Hücre konsantrasyonu gerekiyorsa, biz, bir vakum cihazı ile filtre 0.2 mikron kullanarak filtreleme hücreleri tercih ediyor. Hücreler daha sonra PBS, uygun başka bir tampon veya görselleştirme önce orta filtre yüzeyinden nazikçe yıkanabilir. Slayt hazırlanması 1. 1X Spizizens% 1 agaroz hazırlayın. 5 ile 0.5 g agaroz ile birlikte 10X Spizizens stok ml 45 ml ddH2O ve eritmek için mikrodalga ekleyin. Sıcak erimiş agaroz pipetle zor ve agaroz pipet girmez pekiştirdi. Agaroz ° C pipet girmek için içinde katılaşabilme olmadan, bir sıcaklık 55-65 ~, bir su banyosu içinde dengelenmiş olmalıdır<html> ucu. 2. % 1 erimiş agaroz Spizizens 20 ul pipet içine çizin. Agaroz çözüm sığ agaroz pedleri oluşturmak için her slayt (15 iyi slaytlar, bkz. Tablo 2) tek bir damla (yaklaşık 1-2 mcL) Pipet. Agaroz hemen pekiştireceğiz. Slaytlar da hücrelerin uygulamaya hemen önce yapılmalıdır. Agaroz pedleri sadece oda sıcaklığında bırakılırsa, onlar birkaç dakika içinde kurutmak ve kullanılamaz hale gelebilir. Agaroz pedleri merkezli ve her doldurmak iyi ama lamel bozmamak için en az yüksekliğe sahip olmalıdır; damlacıkları (baloncuk) gibi çok yüksek ya da örnek de sınır ötesine yayılır, sadece tokatlamak bir Kimwipe iyi temiz ve uygulamak Yeni agaroz ped. 3. Her agaroz pad üstüne dağıtılan slayt, tüm 300 ul örneği (bkz. adım 6) uygulayın. Bu hücre miktarı slaytta her iyi karşılamak için yeterli olmalıdır. 4. Yaklaşık on dakika boyunca oda sıcaklığında (23 ° C) oturup hücreleri yüklü slayt izin verin. Bu adım, agaroz ped hareketsiz görüntüleme için ve hazır olma niyet hücreler için izin verecektir. Bir sıcaklık değişimi 4 gerektiren deneyler yaparken, özel ihtiyaçları için bu sıcaklık, örneğin, değiştirilmesi gerekebilir . 5. Pedleri kendileri çıkarmadan her kuyudan fazla orta aspire edin. 6. Slayt alanı boyunca, yavaşça ve eşit sıkıca basarak, ancak, slayt lamel uygulayın. Kapak kayma, slayt karşı ve kapak kayma slayt üzerinde yükseltilmiş değildir olduğundan emin olmak için kontrol edin. Hücre Görselleştirme 1. Birçok farklı floresans mikroskopları iyi çalışacaktır. 100X immersiyon yağı lens için kritik öneme sahiptir. Simmons Lab kullanır: <ul><li> Olympus BX61 mikroskop 1,45 NA TIRFM 100X immersiyon yağı objektif lens ile donatılmış </li><li> Hamamatsu ORCAR 2 CCD kamera soğutmalı </li><li> Lümen 200 ark metal (Önceki) ışık kaynağı. </li><li> GFP (FITC), filtre uyarma 460-500 ve 510-560 emisyon tespiti için </li><li> FM4-64 (TRITC), filtre, uyarma 510-560 ve emisyon 572-648 tespiti için. </li><li> Görüntüler SlideBook 4.2 (Şekil 1) kullanarak ele geçirildi </li></ul> 2. Hücreleri, beyaz ışık kullanarak odak noktası haline getirin. 3. Her fluorofor için uygun filtreleri kullanarak görüntü yakalayın. Temsilcisi Sonuçlar Temsilcisi görüntüleri (Şekil 1). FM4-64 boyama 4-7 parlak ve net olmalıdır GFP odakları, iyi tanımlanmış olmalıdır. GFP veya membran görüntüleri herhangi bir veri kaybı olmadan sunulacak en yüksek kalitede görüntü için izin vermek için herhangi bir renk ile sözde renkli olabilir. <img src="/files/ftp_upload/1736/1736fig1.jpg" alt="Şekil 1" /> Şekil 1: B. Temsilcisi GFP görüntüleri FM4-64 membran boyanması kırmızı ile gösterilen ise subtilis translasyonel füzyon proteinleri GFP proteinler, yeşil gösterilmiştir. Beyaz ölçek çubuğu 3 mikron gösterir (A) bisiklet, GFP ilgili genotip: bisiklet, GFP (spc), amyE: Pspac mutL (kedi)] 600 mg / ml 2-aminopurine ve 1 mM IPTG varlığı. FM4-64 sinyal bisiklet, GFP odakları daha net bir şekilde göstermek amacıyla sunulan değildir (B) MutL GFP [ilgili genotip: mutL: mutL-GFP (spc)]. 2-aminopurine varlığı (C) DnaX-GFP [ilgili genotip: dnaX: dnaX-GFP (spc)]. (D) reca-GFP ilgili genotip: Reca: reca-GFP (spc)] varlığı 20 ng / ml mitomisin C (E) TagC-GFP [ilgili genotip: tagC: tagC-GFP (spc) 1 mcg / ml mitomisin C varlığı] <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tedavi / konsantrasyon veya miktarı </td><td> Fonksiyon </td><td> Inkübasyon süresi </td></tr><tr><td> Mitomisin C (MMC) [20 - 150 ng / ml] </td><td> DNA ajan alkilleyici </td><td> 1 saat </td></tr><tr><td> 2-aminopurine (2-AP) [600 mg / ml] </td><td> uyumsuzluğu indükleyici </td><td> 1 saat </td></tr><tr><td> Hidroksiüre (HU) </td><td> dNTP tüketir </td><td> 3 saat </td></tr><tr><td> ultraviyole ışınları (UV) 20 J/m2 </td><td> Timin-timin dimerleri ve 6-4 photoproducts </td><td> 20 saniye, genellikle kaynağına bağlı olarak değişir. </td></tr><tr><td> griler (iyonizan radyasyon) 5 100 Gy </td><td> Çift iplikli sonları, tek zincirinde kırılmalara ve baz hasarı siteleri </td><td> kaynağına bağlı olarak değişir. </td></tr></tbody></table>&quot;DNA&gt; Tablo 1 Özet zarar ve uyumsuzluğu indükleyici tedavileri <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reaktifin Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Multi slayt, 15-iyi </td><td> MP Biyomedikal </td><td> 6041505E </td><td></td></tr><tr><td> Mikroskop Kapak Camlar </td><td> Balıkçı </td><td> 12-544-B </td><td></td></tr><tr><td> Agaroz </td><td> Balıkçı </td><td> BP160-500 </td><td></td></tr><tr><td> Mitomisin C </td><td> Sigma </td><td> M0503-2MG </td><td> mutagen, eldiven </td></tr><tr><td> 2-Aminopurine </td><td> Sigma </td><td> A3509-250mg </td><td> eldiven </td></tr><tr><td> Hidroksiüre </td><td> Sigma </td><td> H8627-25G </td><td> eldiven </td></tr><tr><td> FM4-64 </td><td> Invitrogen </td><td> T13320 </td><td> ışığa karşı hassastır </td></tr></tbody></table> Tablo 2. Özel reaktifler ve ekipman listesi:

<ol>
	<li>Hardwood, C. R., Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biological Methods for Bacillus. , (1990).</li><li>Berkmen, M. B., Grossman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+organization+of+the+Bacillus+subtilis+replication+cycle.">Spatial and temporal organization of the Bacillus subtilis replication cycle.</a> Mol. Microbiol. 62, 57-71 (2006).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clp+and+Lon+proteases+occupy+distinct+subcellular+positions+in+Bacillus+subtilis.">Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis.</a> J. Bacteriol. 190, 6758-6768 (2008).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+is+required+for+the+RecA+localization+response+to+DNA+damage+in+Bacillus+subtilis.">Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U S A. 104, 1360-1365 (2007).</li><li>Simmons, L. A., Davies, B. W., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=b+Clamp+Directs+Localization+of+Mismatch+Repair+in+Bacillus+subtilis.">b Clamp Directs Localization of Mismatch Repair in Bacillus subtilis.</a> Mol. Cell. 29, 291-301 (2008).</li><li>Simmons, L. A. <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+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction.">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J. Bacteriol. 191, 1152-1161 (2009).</li><li>Smith, B. T., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+mismatch+repair+in+bacterial+cells.">Visualization of mismatch repair in bacterial cells.</a> Mol. Cell. 8, 1197-1206 (2001).</li></ol>

Deneme ve yanılma her suşu için yüksek kaliteli görüntüler için maruz kalma koşullarını bulmak için gereklidir; 100 2000 ms pozlama GFP (FITC) ve FM4-64 için uygun iken, 1 milisaniye beyaz ışık görüntüler için uygun olduğunu bulmak (TRITC ) görüntüleri. Pozlama süresi kullanılan görüntüleme cihazları bağlı olarak değişecektir. Biz, aynı slaytta birden fazla suşları mevcuttur ped ped sınır hücrelerinin kalite ve difüzyon gerginlik farklılaşma karmaşık hale getirebilir basit g?...

Özel çözüm tarifleri: 1 10x S7 50 tuzları 0,5 M MOPS 100 mM Amonyum Sülfat 50 mM potasyum fosfat mono Sterilize Filtresi ve depolama önce sarın S7 folyo ile 50 medya 100x Metaller 0.2 M MgCl 2 70 mM CaCl 2 5 mM MnCl 2 0.1 mM ZnCl 2 100 mcg / mL Tiamin HCl 2 mM HCl 0.5 mM FeCl 3 * dH 2 O son hacim * FeCl 3 yağış önlemek için, son ilave edilmelidir. Filtre Sterilizasyon sonrasında, folyo sarın 100x Metal çözüm. S7 50 medya 1x S7 50 tuzları 1x Metal 1% Glikoz 0.1% Glutamat 40 mcg / ml Triptofan 40 mcg / ml Fenilalanin H 2 O son hacim distile 10x Spizizens (gram / ​​L) 151.4 mM Amonyum Sülfat (20g / L) 803,8 mM potasyum fosfat mono (140g / L) 440,9 mM potasyum fosfat dibazik (60g / L) 34,0 mM Sodyum Sitrat (10g / L) 16.6 mM MgSO 4 (2 g / L) dH 2 O son hacim Sterilize filtreleme

imaging mismatch repair and cellular responses to dna damage in bacillus subtilis

Hem prokaryot ve ökaryotlarda DNA hasarı karmaşık bir dizi fizyolojik değişiklikler ile yanıt verir. Gen ekspresyonu, mevcut proteinlerin yeniden dağıtımı ve yeni protein kompleksleri montaj değişiklikler DNA lezyonları ve eşleşmeyen DNA baz çifti çeşitli teşvik edilebilir. Floresan mikroskopi DNA lezyonları ve karmaşık hücre içi mimari canlı bir hücre içinde DNA replikasyon durumunu izlemek için bu ve diğer tepkiler görselleştirilmesi ve ölçülmesi için güçlü bir deney aracı olarak kullanılmıştır. , Floresan muhabiri proteinleri ve DNA replikasyonu ve tamiri makine bileşenleri arasında Translasyonel füzyonlar ipuçlarını belirlemek için kullanılan hedef DNA onarım proteinleri cognate lezyonlar<em&gt; In vivo</em&gt; Ve bu proteinlerin bakteri hücrelerinin içinde nasıl organize edilir, anlamak için. Buna ek olarak, DNA hasarı indüklenebilir sahiplerine bağlanan transkripsiyonel ve translasyonel füzyonlar nüfus içinde hücre genotoksik stres yanıtları aktif olduğunuz ortaya koymuştur. Bu derlemede, floresan mikroskobu kullanarak görüntü, DNA onarım ve tek bir bakteri hücrelerinin DNA replikasyonu kompleksleri montaj için detaylı bir protokol sağlar. Özellikle, bu çalışmanın görüntüleme uyumsuzluğu onarım proteinler, homolog rekombinasyon, DNA replikasyonu ve SOS-indüklenebilir bir protein üzerinde duruluyor<em&gt; Bacillus subtilis</em&gt;. Burada açıklanan tüm prosedürler bakteri türlerinin çeşitli görüntüleme protein kompleksleri kolayca mükellef bulunmaktadır.

Watch this Scientific Journal Video about Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis at JoVE.com

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Ayrıntılı bir protokol görüntüleme için DNA onarım komplekslerin gerçek zamanlı oluşumu açıklanmıştır<em&gt; Bacillus subtilis</em&gt; Hücreler.

Görüntüleme Uyuşmazlığı DNA Hasar Onarım ve Hücresel Yanıtları Bacillus subtilis</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

imaging-mismatch-repair-cellular-responses-to-dna-damage-bacillus

Research

JoVE Journal

Biology

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

10.9K Görüntüleme.  University of Michigan-Ann Arbor. Ayrıntılı bir protokol görüntüleme için DNA onarım komplekslerin gerçek zamanlı oluşumu açıklanmıştır Bacillus subtilis Hücreler.

Video: Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Biyoloji

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

Whole Sırasının NGS Analiz için bir transpozaz tabanlı Technology tarafından gDNA Zenginleştirme<em&gt; BRCA1</em&gt;<em&gt; BRCA2</emDNA Hasarı Onarım ilgilendim&gt; ve 9 Genler

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Elektron Spektroskopik Görüntüleme Kombinasyonunun miniSOG Etiketli DNA Onarım Proteinler Görselleştirme (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Kronik sonra fare dalak DNA hasar ve Onarım Ölçme<em&gt; In Vivo</em&gt; Beta ve gamma-Radyasyonun Çok Düşük Doz Maruz Kalma

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Sıçan Karaciğer DNA Strand Breaks ve Oksidatif DNA Hasar Ölçüm Vivo Alkali Comet Assay ve Enzim-modifiye Alkali Comet Tahlili

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Zararlı etkileri, düşük basınç plazma sterilizasyon Bacillus subtilis sporlar kullanarak canlı cep mikroskobu sağkalıma soruşturma

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Bacillus subtilis tek hücreli mikrosıvısal analizi

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Endononülaz Bazlı Meydana Özgü DNA Hasarlarını Görselleştirme ve Ölçme

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In Vitro E3 Ubiquitin Ligase fonksiyonunun analizi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...