Name: electroporation of functional bacterial effectors into mammalian cells
Uploaded: 2015-01-19T13:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1. Advance hazırlayın <ol><li> 37 ° C sıcak bir steril fosfat tamponlu tuz (PBS). </li><li> % 10 fetal sığır serumu (FBS) ile desteklenmiş Eagle Medium (DMEM) ve Minimal Essential Medium (MEM) Sıcak Dulbecco modifikasyonu, 100 lU / ml penisilin ve 100 ug / ml streptomisin, 37 ° C arasındadır. Not: Bunlar sırasıyla RAW ve HeLa hücreleri için normal Büyüme Medya (NGM) temsil eder. </li></ol> Hücreler 2. Hazırlık <ol><li> NGM içinde% 70-90 confluency RAW 264.7 hücreleri büyümek. <ol><li> Nemlendirilmiş% 95 hava /% 5 CO2 atmosferinde, 37 ° C&#39;de hücreler muhafaza edin. </li></ol></li><li> NGM içinde% 70-90 confluency HeLa hücreleri büyür. <ol><li> Nemlendirilmiş% 95 hava /% 5 CO2 atmosferinde, 37 ° C&#39;de hücreler muhafaza edin. </li></ol></li><li> Toplanmadan önce steril PBS ile bir kez, hücre tekli tabakası yıkayın. </li><li> Steril konik bir tüp içinde önceden konfluent hücreler toplanır. <ol><li> Yavaşça RAW hücreleri kazıyınPBS içinde, bir lastik policeman ile tekrar pipetleme hücre toplamalardan dağıtmak için. </li><li> Görsel muayene kültür yüzeyinden ayrışma gösterene kadar% 0.25 tripsin solüsyonu ile HeLa hücreleri ayırmak. Örneğin, T-75 balonuna için 2-3 ml kullanılır. Tripsin çözüm tüm büyüme yüzeyini kaplayan sağlamak için buna göre ses seviyesini ayarlayın. <ol><li> NGM,% 10 FBS içeren ayrılma reaksiyonu söndürün. </li><li> Hücre toplamalardan dağıtmak için tekrar pipetleme ile, tripsin için NGM en az iki hacim kullanın. </li></ol></li></ol></li><li> Hafif 4 dakika boyunca 900 x g&#39;de santrifüj ile pelet hücreleri. </li><li> Steril PBS kullanarak tripsin / söndürme çözüm olarak aynı hacimde süspanse. </li><li> Hemasitometre veya parçacık sayacı kullanarak hücreleri saymak. </li><li> Hafif 4 dakika boyunca 900 x g&#39;de santrifüj ile pelet hücreleri. </li><li> 5,5 x 10 6 6 10 x 6.0 hücre / ml PBS yeterli hacimde süspanse. Not: Örneğin, bir T-75 şişe approxim verecektirulaştırılması 7.5 x 10 6 hücre 29. </li><li> Elektroporasyon kadar buz üzerinde hücre süspansiyonu tutun. </li></ol> Elektroporasyon 3. Hazırlık <ol><li> Buz Öncesi soğuk elektroporasyon küvetler (0.4 cm boşluk). </li><li> Elektroporasyon aparatı açın ve 0,3 kV gerilim ayarlayın. NOT: Kapasite ve direnç (üretici tarafından 10 iF ve 600 Ω ayarlı) bizim elektroporatörü ayarlanabilir ayarları değildi. </li><li> NGM ile geri kazanım plakaları, doldurun ve 37 ° C de, nemlendirilmiş bir% 95 hava /% 5 CO2 atmosferinde dengeye </li></ol> 4. Elektroporasyon <ol><li> Önceden soğutulmuş küvet içinde hücre süspansiyonu 400 ul koyun ve küvetin (ug / ml 50 ug) seçilen proteinin 20 ug ekleyin. </li><li> Flick küvet hafifçe ~ 10 kez hücrelere zarar vermeden karıştırın. Not: küvet de iyice karıştırın birkaç kez ters olabilir, ancak yukarı pipetlemeyin yok ve aşağı ya da vorteks hücreleri zarar vermemek için. </li><li&gt; Kağıt havlu veya diğer silecek ile küvetin kuru dış Elektroporatör elektrik arkı önlemek için. </li><li> 1.5-1.7 msn 0.3 kV electroporate örnek. Not: Bu, bu çalışma için tipik oldu. </li><li> Hemen elektroporasyon fiske küveti sonra yavaşça ~ 10 kat iyice karıştırın. </li></ol> Hücreler 5. Kaplama <ol><li> Hazır olana kadar buz üzerinde Electroporated hücreleri ile mağaza küvet kurtarma plakalarda yer. </li><li> En mansap analizler için, yabancı efektör protein kaldırmak için önceden ısıtılmış NGM ile hücreler 1x yıkayın. <ol><li> Analiz için hücreleri kaldırmak ve NGM 3-5 ml askıya. </li><li> 4 dakika boyunca 900 x g&#39;de santrifüj ile pelet hücreleri. </li><li> Istenen plaka boyutuna (örneğin 35 mm tabak için 2 mi) için NGM yeterli hacmi içinde süspanse. </li></ol></li><li> Mansap analizi için hücrelerin uygun miktarda çıkarın. <ol><li> Örnek 1: mikroskopi analizi için cam alt yemekleri içine plaka. </li><li> Örnek 2: levha intBöyle afinite saflaştırma gibi protein analizi için o hücre kültürü plastik. </li></ol></li><li> Hücrelerin en az 4 saat 37 ° C&#39;de nemlendirilmiş% 95 hava /% 5 CO2 atmosferde dengelenmiş plakalarda kurtarmak için izin verin. </li></ol> 6. Mikroskopi Analizi 6.1) Sabitleme / İmmünofloresan Boyama <ol><li> Yıkama hücreleri 4 saat iyileşme döneminden sonra steril PBS ile 1x. </li><li> Oda sıcaklığında 2 dakika boyunca% 100 metanol hücreleri saptamak. Tamamen hücreleri (örneğin 2 ml 35 mm çanak için) karşılamak için yeterli metanol kullanın. </li><li> Steril PBS ile yıkayın 3x. </li><li> 15 dakika boyunca PBS içinde% 0.4 Triton X-100 ile hücrelerin geçirgenliği. Örneğin, bir 35 mm çaplı levha 1 ml kullanılır. <ol><li> Epitop ile hedef proteinin konumuna göre, Triton X-100 geçirgenliği ve gücü uzunluğunu ayarlayın. Not: En iyi sonuçları ancak yukarıdaki koşullar çoğu c yeterli olmalıdır, ampirik her hedef için belirlenen gerekecektirytosolic hedefler. </li></ol></li><li> Oda sıcaklığında 1 saat için PBS içinde% 5 sığır serum Albumin (BSA) ile bloke edin. Örneğin, bir 35 mm çaplı levha 1 ml kullanılır. </li><li> PBS ile yıkayın 3x. </li><li> 4 ° C&#39;de bir gece boyunca antikor bağlayıcı çözelti içinde birincil antikor (PBS içinde% 0.1 Triton X-100 ve% 1 BSA) inkübe &#160; Hafifçe çalkalanarak ° C. Örneğin, bir 35 mm çaplı levha için 0,5 ml kullanılır. <ol><li> Antikor seyreltme için üreticinin tavsiyelerini uygulayın. Not: 200 seyreltme: PKN1 antikoru 1 kullanıldı olurken, 1000 seyreltme Örneğin, streptavidin bağlayıcı peptit etiketi (SBP-etiket) antikoru 1 kullanılmıştır. </li></ol></li><li> PBS ile yıkayın 4x. </li><li> Işıksız bir ortamda oda sıcaklığında 1 saat süre ile antikor bağlama çözeltisi içinde uygun floresan konjuge sekonder antikor ile inkübe edin. <ol><li> Örneğin, oda sıcaklığında 1 saat süre ile Alexa 488 veya Alexa 647 kullanır. <ol><li> Karıncanın üreticinin önerilerini izleyinibody seyreltme. (Örneğin, yaklaşık 1: 1000, bu çalışma için). </li></ol></li><li> Örneğin, gerekli olan diğer lekeler ekleme, 5 uM Buğday agglutinnin (WGA) ile oda sıcaklığında 1 saat süre ile, üreticinin tavsiyesine göre Alexa 647 veya DAPI birleştirilir. </li></ol></li><li> Görüntü hazır olana kadar ışıktan korundu PBS ile 4 ° C &#39;de deposu ile yıkayın 5x. </li></ol> 6.2) Konfokal Mikroskopi ve Görüntü Analizi <ol><li> 63x immersiyon yağı hedefi kullanarak bir ters konfokal mikroskop görüntü örnekleri. <ol><li> 492-542 nm arasındaki bant genişliği emisyonu ile argon lazer 488nm hattını kullanarak görüntü yeşil kanal. </li><li> 640-718 nm arasındaki bant genişliği emisyonu ile 633 nm diyot lazer kullanarak görüntü kırmızı kanalı. </li><li> 407-453 nm arasındaki bant genişliği emisyonu ile 405 nm diyot lazer kullanarak görüntü mavi kanal. </li><li> Z-yığınları hücresel hacmi bütününü kapsayan çoklu kanal emin olun. </li></ol></li><li> Uygun görüntü işleme yazılımı ile Süreç görüntüleri. </li></ol> 7. Affinity Arıtma <ol><li> 4 saat iyileşme süresinden sonra 4 ° C&#39;de iki kez PBS ile elektroporasyona hücreleri yıkayın. </li><li> Ile lizleyin ~ liziz tamponu buz üzerinde (PBS içinde proteaz inhibitör kokteyli ve fosfataz inhibitörü ile% 1 Triton X-100) ve 1.0 mi. Üretici talimatlarına göre inhibitörleri kullanın. Not: Örneğin, her ikisi de bu çalışmada kullanılan fosfataz ve proteaz inhibitörleri 100x de temin edilmiştir, ancak diğer formülasyonlar eşit derecede iyi çalışmasıdır. </li><li> Derhal lastik polis hücreleri kazıyın ve konik tüpler içine toplamak. </li><li> Güçlü döndürülerek ve sonikasyon ile lizleyin. Not: Diğer lizis yöntemleri eşit olarak iyi çalışabilir, ancak verimlilik ampirik tahlil gereksinimleri uygunluğu tespit edilecek gerekecektir. <ol><li> Aralıklı vorteks 3 x 30 sn sonikasyon. </li></ol></li><li> 4 ° C&#39;de 10 dakika boyunca 10,000 x g&#39;de santrifüjleyin toplamakHücresel enkaz ve çözünmeyen agrega; süpernatant kaydedin. </li><li> Sonu aşırı uç rotasyon ile bir gece boyunca 4 ° C &#39;de Streptavidin agaroz reçinesi süspansiyonuna 50 ul ile elektropore edildi hücre lizatının eşit hacimleri (~ 1.0 mi) birleştirin. Not: Bu reçine olan streptavidin bağlama peptidi afinite etiketi ile elektropore protein (ve ilişkili kompleksler) yakalar. </li><li> 2 dakika için 2.500 xg&#39;de Santrifüj ve süpernatant atın. </li><li> 40 yatak hacmi (~ 1 mi) iki kez PBS ile yıkayın. </li><li> 30 ul 4x LDS yükleme tamponu (141 mM Tris baz,% 2 LDS,% 10 gliserol, 0.51 mM EDTA, 0.22 mM SERVA Mavi G kırmızı 0.175 mM fenol, pH 8.5), 20 ul diH 2 O ve 1 ul ekle bir hacim sağlayan, 0.5 M tris (2-karboksietil) fosfin (TCEP); boncuk içinde kalması beklenir. </li><li> 10 dakika boyunca 95 ° C de ısı ve buz üzerinde soğutulur. </li><li> Sıkma 5 dakika boyunca 4 ° C &#39;de&gt; 10,000 xg. </li><li> Süpernatant toplayın. </li><li> Uygun antikorlar Not kullanarak batı leke gerçekleştirin: OYeniden anti-PKN1 birincil antikor kullanılır. </li></ol>

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D., 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=Identification+of+GtgE,+a+novel+virulence+factor+encoded+on+the+Gifsy-2+bacteriophage+of+Salmonella+enterica+serovar+Typhimurium.">Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium.</a> Journal of Bacteriology. 184, 5234-5239 (2002).</li><li>Niemann, G. S., 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=Discovery+of+novel+secreted+virulence+factors+from+Salmonella+enterica+serovar+Typhimurium+by+proteomic+analysis+of+culture+supernatants.">Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.</a> Infection and Immunity. 79, 33-43 (2011).</li><li>Spano, S., Liu, X., Galan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+targeting+of+Rab29+by+an+effector+protein+distinguishes+the+intracellular+compartments+of+human-adapted+and+broad-host+Salmonella.">Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 18418-18423 (2011).</li><li>Haraga, A., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., 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=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., 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=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Patojen bakterilerin salgılanan efektörleri konak hücre ortamında çalışabilmesi için geliştiğini ve böylece konak içinde in situ bunları incelemek yararlı olacaktır. Konakçı hücrelere özel ilgi efektörlerinin giriş ilgili patojen-konakçı etkileşimleri bakteriyel proteinlerden müdahalesi olmaksızın tek başına ele sağlar. amaç, böylece transfeksiyon ya da transdüksiyon ile bağlantılı bazı zorluklar kaçınarak ökaryotik konakçı hücrelere bakteriyel efektör proteinler kat?...

Bir çok Gram-negatif bakteriler, doğrudan konakçı hücreler 1-5 içerisine (efektör olarak anılacaktır) hastalık oluşturma ilişkili proteinler enjekte özel salgılama sistemleri kullanılmaktadır. Ev sahibi dokunulmazlık baskılanması, hücre iskeleti değişiklikler, hücre içi ticareti ve sinyalizasyon, transkripsiyon değişikliklerin modifikasyonu ve ev sahibi proteasome değişiklikler 6-9: Bu efektörleri dahil olmak üzere biyolojik fonksiyonları geniş bir yelpazesi var. Bazı efektörlerinin işlevleri ancak ev sahibi hedefleri ve diğerleri biyokimyasal eylem (ler) belirlenecek kalır bilinmektedir. Vahşi tip ve yeniden birleştirici bakteriyel enfeksiyonların karşılaştırılması hücre içi efektör hastalık oluşturma mekanizmaları incelemek için geçerli bir yaklaşım olsa da, konakçı hücre içerisine bağımsız bir efektör tanıtmak için sıklıkla yararlıdır. Böylece, konakçı hücreler bağlamında bakteriyel efektör proteinler katma ve karakterize edilmesi için basit bir yöntem arzu edilmektedir. Deneysel analiz wi BasitleştirmeTek bir efektör kritik inci diğer efektörler karşı ya da gereksiz işlevlere sahip olabilir. Bu basitleştirme gerçekleştirmek için, araştırmacılar, daha önce kazıma 12,13 yükleme viral transdüksiyon 10, mikroenjeksiyon 11 de dahil olmak üzere birçok farklı yöntemler ile hücrelere makromolekülleri getirmiştir, kimyasal olarak teşvik mikroenjeksiyon 14, özel bir protein &quot;transfeksiyon&quot;, reaktif 15, kalsiyum fosfat çöktürme ile hücre füzyonu 16 ve elektroporasyon 17-20. kişiye moleküller, hücre içi hedefleri 21,22 protein, hücre-geçirgen olmayan boyalar ve antikorlar, DNA, RNA ve RNAi türler dahil olmak üzere nükleik asitler arasında değişir. Bazı yöntemler dahil edilebilir makromolekül Çeşidi dahil kısıtlamaları vardır ve özellikle alt-analizler, yüksek hücresel toksisite, etki zarar mekanizmaları, düşük etkililik ve giriş verimliliği ile sınırlı olabilir. Transfeksiyon, bir ofteaynı zamanda makrofajlar ve birincil hücre gibi uygun bir ev sahibi hücre tipi, transfeksiyon karşı özellikle dirençlidir sınırlama uğrar, memeli hücrelerinde bakteri genleri ifade etmek için bir yöntem, n-kullanılmıştır. Bunun ötesinde, yabancı DNA&#39;nın sokulması üzerine üretilen bakteriyel protein seviyelerini kontrol etmek zordur. Çok iş bilinen bir laboratuvar tekniği olarak bakteriyel ve memeli yaratıkların hücrelerine nükleik asit elektroporasyon kurdu; Bununla birlikte, fizyolojik koşullar altında, hücre içine proteinlerin verilmesi için en iyi yöntem devam etmekte olan bir araştırma vardır. Protein transfeksiyon üzerindeki raporlar umut vericidir, ama pahalı reaktifler ve optimizasyon gerektirir. minimum maliyet ile hücre hedefleri çeşitli içine potansiyel toksik bakteri efektörler tanıtmak arzusu in vivo bu proteinleri eğitim için bir yöntem olarak elektroporasyon dikkate götürdü. Protein elektroporasyon 23-25 ​​bir araya geldi iseAyrıca elektro-transfeksiyon veya elektro-enjeksiyon 26 olarak bilinen electropermeabilization üzerinden canlı hücreler içine proteinleri tanıtmak hod. Bu teknik, hücre zarlarında gözenekleri oluşturmak için yüksek yoğunluklu elektrik darbeleri kullanır. Bu geri dönüşümlü gözenekler normal hücre içi alandan dışlanan makromoleküllerin hücreye girmesine izin. Dış elektrik alanının çıkarılması üzerine, zar, hücre gözenekler 27,28 geçirilir molekülleri korumak için izin kapatın olabilir. Standart bir Electroporator sürekli fare makrofaj-benzeri hücreleri ve insan epitel hücreleri hem de içine bakteri efektör tanıtmak için bu çalışmada kullanılmıştır. yöntem hücresel canlılığı hiçbir kayda değer azalma ile, hızlı, verimli ve ucuz. kişiye proteinler immünofloresan mikroskopi ile görüntülenmiştir veya fonksiyonel deneyleri için de kullanılabilir. Bu aynı zamanda, bir toksik-olmayan, standart olarak yeşil floresan proteini (GFP) ile gösterilmiştirİki Salmonella efektör proteinler, SspH1 ve GtgE. Bu ökaryotik konakçı hücrelerde bu bakteriyel hastalık oluşturma proteinleri ve işlevlerin çalışması için repertuarında ek bir araç olarak, protein elektroporasyon önerilmektedir.

<table><tbody><tr><td>0.25% Trypsin-EDTA Solution</td><td>Cellgro</td><td>25-050-Cl</td><td></td></tr><tr><td>0.4 cm Gap-disposable electroporation cuvettes</td><td>Bio-Rad</td><td>165-2088</td><td></td></tr><tr><td>100% Methanol</td><td>Any</td><td>N/A</td><td>Flammable, Toxic</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4919</td><td></td></tr><tr><td>Cell Counting Apparatus - Hemocytometer or Coulter Counter</td><td>Beckman Coulter</td><td>Model Z1</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Any</td><td>N/A</td><td>Humidified 95% air/5% CO&lt;sub&gt;2 &lt;/sub&gt;atmosphere at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Cell Culture Plastic</td><td>Any</td><td>N/A</td><td>Cell culture flasks/plates, pipets, tubes, rubber policeman</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&amp;rsquo;s Medium (DMEM)</td><td>Cellgro</td><td>10-013</td><td>Warm to 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Electroporator</td><td>Bio-Rad</td><td>E. coli Pulser</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Cellgro</td><td>35-016-CV</td><td></td></tr><tr><td>Fluorescent confocal microscope&amp;nbsp;</td><td>Ziess</td><td>Model&amp;nbsp; LSM 710&amp;nbsp;</td><td></td></tr><tr><td>Glass Bottom Dishes for Microscopy</td><td>Wilco Wells</td><td>HBSt-3522</td><td></td></tr><tr><td>HALT Protease Inhibitor Cocktail</td><td>Pierce</td><td>78430</td><td>Corrosive, Toxic</td></tr><tr><td>HeLa Cell Line</td><td>ATCC</td><td>ATCC CCL-2</td><td></td></tr><tr><td>LDS 4X Loading Buffer</td><td>Invitrogen</td><td>NP0007</td><td></td></tr><tr><td>Minimal Essential Medium (MEM)&amp;nbsp;</td><td>Cellgro</td><td>10-010</td><td>Warm to 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Cellgro</td><td>30-002-Cl</td><td></td></tr><tr><td>RAW 264.7 Cell line</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>Primary Antibody Against Target of Interest</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Secondary Antibody Conjugated to Fluorophore</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Any</td><td>N/A</td><td>Chill to 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Sterile Phosphate Buffered Saline</td><td>Any</td><td>N/A</td><td>Warm to 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Streptavidin Agarose Resin Suspension&amp;nbsp;</td><td>Pierce</td><td>20353</td><td></td></tr><tr><td>Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>TCEP</td><td>Sigma-Aldrich</td><td>646547</td><td>Corrosive, Toxic</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8585</td><td>Irritant, Toxic</td></tr></tbody></table>

electroporation of functional bacterial effectors into mammalian cells

canlı hücrelerin bağlamında protein etkileşimleri çalışma lokalizasyonu, dinamikleri ve etkileşim ortakları hakkında kritik bilgiler üretebilir. Bu bilgiler, evsahibi-patojen etkileşimlerinin bağlamında özellikle değerlidir. Birçok patojen proteinleri, hücre içi bir ortam içinde konak bağışıklık sistemi ve hayatta kalma kaçırma sağlayan, bu gibi bir şekilde çeşitli konak hücreleri içinde çalışır. Bu patojenin protein konukçu hücre etkileşimini incelemek için çeşitli yaklaşımlar sık dahil olmak üzere kullanılır: in vivo enfeksiyon transfeksiyon ya da transdüksiyon ile etiketli veya mutant protein ya da patojen genlerin katılması eksprese eden bir suşu ile. Bu yaklaşımların her biri avantajları ve dezavantajları vardır. Bu direkt olarak hücrelere ekzojen proteinleri katılması için bir araç çalıştı. Elektroporasyon yaygın hücrelere nükleik asitlerin dahil edilmesi için kullanılabilir, ancak biyo-fiziksel temeli tam olarak aynı olmasına rağmen daha nadir proteinlere uygulanmıştır.Standart bir Electroporator memeli hücrelerine afinite etiketli bakteriyel efektör tatbik etmek için kullanılmıştır. İnsan epiteliyal ve fare makrofaj hücreleri, geleneksel metodlarla kültürlenmek sureti ile ayrılır ve ilgi dahilindeki bir eksojen bakteriyel patojenin proteini (örneğin, Salmonella Typhimurium GtgE) ile 0.4 cm boşluk elektroporasyon küvetler içine yerleştirilmiştir. Elektroporasyon (0.3 kV) ve kısa (4 saat) geri kazanım süresinden sonra, hücre içi protein, flüoresan olarak afinite etiketi ile protein etiketleme ve konfokal mikroskopi ile mekansal ve zamansal dağılımının incelenmesi ile doğrulanmıştır. Elektroporasyona protein aynı zamanda hücre içinde işlevsel ve doğru hücre içi ticareti ve protein-protein etkileşimi yeteneğine sahip olduğu gösterilmiştir. Ekzojen proteinler hücrelerin yüzeyi üzerinde birikme eğiliminde iken, elektroporasyona örnekler sadece kuluçkalama için hücre içi efektör konsantrasyonda büyük artışlar vardı. protokol ap yapılacak basit ve yeterince hızlıhücre içi hedefleme ve hastalık oluşturma proteinlerin işlevine de dahil olmak üzere, konakçı hücrelerde patojen proteinlerinin yüksek verimli bir karakterizasyon için izin arallel moda.

Kavramının bir ilk kanıtı olarak, saflaştırılmış yeşil flüoresan proteini başarıyla elektroporasyon ile memeli hücrelerine dahil edilmiştir. GFP, yaklaşık 27 kD molekül ağırlığına sahip protein özel genellikle önemli hücresel toksisite olmaksızın moleküler biyoloji aracı olarak (normal olarak plasmid DNA ifade edilen), memeli hücreleri içine dahil edilir. HeLa hücreleri (Şekil 1A) kuluçkalanmıştır veya floresan GFP sinyali kontrol etmek için imüno-konfokal mikrosk...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Memeli Hücreleri içinde fonksiyonel Bakteriyel Efektör elektroporasyonu

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.0K Görüntüleme.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

Video: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Mikobakteriler, Elektroporasyon

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Biyoloji

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Memeli İskelet Kaslar kullanarak DNA Transfeksiyon In Vivo Elektroporasyon

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Mühendislik Hücre geçirgen Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Soruşturma memeli Axon rejenerasyon: Vivo Elektroporasyon yetişkin fare Dorsal kök gangliyon

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Nörobilim

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Elektrokompetan Hazırlanması için hızlı Protokolü<em&gt; Escherichia coli</em&gt; Ve<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Fare Hipokampal Uyarıcı ve Sınıfa Özgü inhibitör Nöronların Farklı Organotipik Dilim Kültürü boyunca Tek Hücreli Elektroporasyon

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Gap Kavşak Sınavı Fonksiyonel Testi; İndiyum-Kalay Oksit Yapışık hücrelerin elektroporasyonu

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Uyuyan Güzel Transpozon Sistemi Kullanılarak Primer İnsan Pigment Epitel Hücrelerinin Elektroporasyon Tabanlı Genetik Modifikasyonu

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

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Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

Plazmid DNA'sının Fare İskelet Kasına Elektroporasyonu

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA. In nature, transformation can occur in certain types of bacteria. In molecular biology, however, transformation is artificially induced through the creation of pores in the bacterial cell walls. Bacterial cells that are able to take up DNA from the environment are called competent cells. Electrocompetent cells can be produced in the laboratory and transformation of these cells can be achieve via the application of an electrical field that creates pores in the cell wall through which DNA can pass.
The video explains the equipment used in electroporation such as an electroporator and electroporation cuvette. The video also goes through a step-by-step procedure about how to create electrocompetent cells and electroporate cells of interest. 
Prediction of the success of a transformation of an experiment, by observing the time constant, as well as the importance of removing salt from the solutions when electroporating, are also mentioned.

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...