Biz yararlı tartışmalar için laboratuarımızda tüm üyelerine teşekkür ederim. Biz yazının eleştirel okuma Joanna Lisztwan ve Christina Gewinner ederim. Bu çalışma BBSRC hibe BB/I021248/1 tarafından desteklenen ve Wellcome Trust hibe 090090/Z/09/ZAH UCL Kanser Enstitüsü&#39;nde Wellcome Trust Araştırma Kariyer Geliştirme akademisyendir edildi.

1. PcDNA6_TetR_IRES_blast klonlanması <ol><li> Şekil 3 &#39;de gösterildiği üzere, kısıtlama enzimleri xba I ve NCO bir gen ve Tetr blasticidin direnci (BlastR) gen promoter çıkarmak için birlikte plazmid pcDNA6/TR (V1025-20, Invitrogen) kısmi bir özet gerçekleştirir. 4.6 kb vektör fragmanı ayırın. </li><li> Tetr geninden poliadenilasyon sekansı kaldırmak için, bir Xho I site hemen Tetr bir stop kodonu cDNA 5&#39;end de muhafaza xba I site süre sonra cDNA, PCR ile getirmektedir. PMigR1_TetR plazmid üretmek için kısıtlama enzimlerinin xba I ve Xho I ile pMigR1 17 içine sonuçtaki fragman Subclone. </li><li> TetR_IRES fragmanı serbest restriksiyon enzimleri xba I ve Astsubay ben pMigR1_TetR vektör sindirmek. 1.25 kb ekleme parçası ayırın. </li><li> 4.6 kb pcDNA6/TR ve 1.25 kb TetR_IRES parçaları Arter. TrYetkili E.coli içine ligasyon ürünü ansform ve standart moleküler biyoloji teknikleri kullanarak istenen pcDNA6_TetR_IRES_blast yapı tanımlar. </li></ol> 2. Hücre Hatları Nesil stabil Tetr ifade <ol><li> Günde 1, Tohum 1x10 Açık standart büyüme ortamı kullanılarak 10 cm doku kültürü plaka başına 6 hücre (örneğin DMEM% 10 FCS ile takviye). Tohum iki levha, pcDNA6_TetR_IRES_blast ile transfeksiyon için, diğeri ise blasticidin seçimi için bir negatif kontrol olarak, diğer. Mümkün olan en düşük geçiş ve tavsiye edilen büyüme ortamında kullanmak için emin olun. </li><li> Günde 2 üzerinde, transfekte üreticinin yönergelerini izleyerek Fugene 6 (E2691, Promega) kullanılarak pcDNA6_TetR_IRES_blast 2 mikrogram ile bir tabak. Ikinci plaka transfekte etmeyin. </li><li> 3. günde, iki tabak için 5 mg / ml blasticidin içeren standart büyüme ortamı ekleyin. Optimum seçimi konsantrasyonu belirli bir hücre hattı için farklı olabileceğini lütfen unutmayın olabilir nönceden belirlenecek eed. </li><li> 4. günde, 10 cm tabak ve 5 mg / ml blasticidin içeren standart büyüme ortamı kullanılarak 1/50 ve 1/100, 1/25, 1/5 de hücreleri bölme. Düzgün untransfected veya transfekte hücreler içeren plakalar etiketlemek için emin olun. Seyreltme adım başına iki tabak hazırlayın. </li><li> Untransfected plakalar üzerinde tüm hücreleri ilk hafta içinde öldüğü sağlanması, günlük hücreleri denetleyin. 2-3 günde bir seçim ortamı değiştirin. </li><li> 1-2 hafta sonra, blasticidin dayanıklı koloniler görünmeye başlaması gerekir. Tek koloniler toplandı olmasını sağlamak için 5-50 ihtiva eden koloniler plakalar seçin. </li><li> Koloniler yaklaşık olarak 5 mm (ya da daha büyük) bir çapa ulaştığı zaman, tek koloniler almak için klonlama silindir kullanılarak, en az 12 bağımsız klon izole eder. 24 iyi doku kültürü plaka ayrı bir kuyuya her klon aktarın. Ne zaman birleşmiş, iyi bir 6-doku kültür plakasının tek tek her bir kuyu gelen hücreleri ayrıldı. </li><li> Kültür cl DevamSeçimi medya olanları. 6-iyi doku kültürü plaka iki tek kuyulara her kuyudan zaman konfluent, split hücreler. </li><li> Her klon test edilmesi için immunblotting sonraki test için başka bir kuyuya confluent hücreleri ve hasat hücreleri de bir dondurma. </li><li> Batı fare monoklonal anti-TET02 antikor (Mobitec GmbH) kullanarak blotting ile Tetr algıla. Bir negatif kontrol olarak ebeveyn hücre hattının bir lizat dahil unutmayınız. </li><li> Yüksek Tetr ifade ile 3 klon seçin ve her klonal kültür genişletin. En kısa sürede, her umut verici klonu stokları dondurun. Son olarak, tetr-salgılayan klonlar ile karşılaştırarak ana hücre soyu ile, ilgilenilen ve moleküler biyolojik hücre parametreleri inceleyin. Ilgi moleküler ve hücre biyolojisi parametrelerinde herhangi bir değişiklik göstermez yüksek Tetr ifadesi ile &#39;en iyi&#39; klon seçin. </li></ol> 3. Pter ve pT-Rex DEST30 Vektörler Pilot Test ExpressSırasıyla ing shRNA veya cDNA, <ol><li> 12 açıklandığı gibi shRNA ekler ile plazmid pter oluşturun. Pt-Rex DEST30 vektör (12301-016, Invitrogen) ve ağ geçidi teknolojisi (Invitrogen) kullanılarak ilgi cDNA içeren Construct. CDNA ifade etmesi halinde, cDNA ile bir etiketin ilave daha sonra ifade edilmiş protein Eksojen olarak tespit kolaylaştıracaktır. </li><li> Geçici seçim transfeksiyon reaktif kullanılarak pter veya pT-Rex DEST30 plazmidler (shRNAs veya ilgi cDNAlarının da ifade) ile Ebeveyn hedef hücre hattı transfekte. ShRNA ifade plazmid test için, yüksek transfeksiyon verimlilik elde edilebilir sağlamak. </li><li> Hasat ilgi gen tükenmesi veya aşırı algılamak için bekliyor, uygun antikorlar kullanılarak immunoblotting için hücreleri ve süreç transfekte. </li></ol> 4. Tetrasiklin-indüklenebilir (Tet-on) pter veya pT-Rex DEST30 Vektörler kullanma shRNAs veya cDNAlann Expression Kararlı Hücre Hatları Üretimi </p&gt; <ol><li> Günde 1, sertifikalı Tetrasiklin serbest serum (; 16000-014 Invitrogen örneğin FBS için) ile desteklenmiş standart büyüme besiyeri kullanılarak 10 cm plaka başına &#39;en iyi&#39; Tetr-ifade klonu tohum 1x10 6 hücre üzerinde. Pter (veya pT-Rex DEST30), ve çift seçimi için negatif kontrol olarak bir plaka ile transfeksiyon için Tohum bir tabak. Mümkün olan en düşük geçiş kullandığınızdan emin olun. </li><li> Günde 2 üzerinde, transfekte Fugene 6 kullanarak pter 2 mikrogram (veya pT-Rex DEST30) ile bir tabak. Ikinci plaka transfekte etmeyin. </li><li> 3. günde, 5 ug / ml blasticidin ve 500 ug / ml zeocin (ya da 1 mg / ml G418) ihtiva eden standart büyüme ortamı ilave edin. </li><li> 4. günde, 5 mg / ml blasticidin ve 500 mg / ml zeocin (veya 1 mg / ml içeren 10 cm tabak ve standart büyüme ortamı kullanılarak 1/50 ve 1/100, 1/25, 1/5 de hücreleri bölme G418). Seyreltme adım başına iki tabak hazırlayın. </li><li> Ilk hafta içinde untransfected tabaklar üzerindeki ölenlerin emin, günlük hücreleri denetleyin.Blasticidin ve zeocin (veya G418) her 3-4 günde içeren standart büyüme ortamı değiştirin. </li><li> 2-3 hafta sonra, blasticidin / zeocin (veya blasticidin/G418) çift dayanıklı koloniler görünmeye başlaması gerekir. Tek koloniler almak için 5 ila 50 koloni içeren plakalarda seçin. </li><li> Koloniler yaklaşık 5 mm (veya daha büyük) bir çapa ulaştığında, tek koloniler almak için klonlama silindir kullanın. En az 24 ayrı klonlar ayırın. 24 iyi doku kültürü plaka ayrı bir kuyuya her klon aktarın. </li><li> Blasticidin (5 ug / ml) ve zeocin (250 ug / ml) ihtiva eden bakım ortamı konsantrasyonları kültür klonlar [G418 ya da (0.5 mg / ml)]. Ne zaman birleşmiş, iyi bir 6-doku kültür plakasının tek tek her bir kuyu gelen hücreleri ayrıldı. </li><li> Her bir klon test edilmesi için, iyi bir 6-kuyulu plakanın kuyularına üç tek bir 6-kuyulu plakanın bir konfluent ayrıldı. Zaman konfluent, iyi biri hücreleri dondurmak. Tetrasiklin test için kalan iki kuyu kullanınE-indüklenebilir ifade. Ikinci da tetrasiklin serbest bırakırken, iyi bir ila 1 ug / ml &#39;lik bir konsantrasyonda tetrasiklin ekleyin. </li><li> Uygun antikorlar kullanılarak immunoblotting için Tetrasiklin, hasat hücreleri ve süreç ile inkübasyondan 24 ila 96 saat sonra. CDNA ifadesinin indüksiyonu için tarama yaparken uzun tetrasiklin tedavisi endojen proteinlerin shRNA aracılı tükenmesi belirlemek için gerekli olacak ise kısa tetrasiklin inkübasyon sürelerinde, tavsiye edilir. </li><li> İstenen tükenmesi / aşırı ekspresyonu ile en az iki klon seçim ve her bir kültür genişletmek. En kısa sürede, her umut verici klonu stokları dondurun. Deneyler için klonlar Tet-büyüyen daima Tetrasiklin serbest serum ve bakım konsantrasyonlarda ilgili seçim antibiyotik ile desteklenmiş ortam kullandığınızdan emin olun. </li></ol>

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Çıkar çatışması ilan etti.

Biz, Tet-on sistemi büyük ölçüde özellikle manipüle gen hücrenin hayatta kalması için gerekli olan zaman idare ve / veya zor olan hücre sistemleri, gen fonksiyonu analizini kolaylaştırmak inanıyoruz. Ayrıca, son derece spesifik Tet-on sistemi ile gen ekspresyonu kontrol etme yeteneği farklı aşamalarında (hücre siklus progresyonu veya iyi tanımlanmış farklılaşma süreçleri sırasında örneğin) genlerin fonksiyonlarının çalışma fırsatı sunuyor. Burada sunulan yöntem araştırmacılar ol...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Sığır fetüs serumu (FBS) </td><td> Invitrogen </td><td> 16000-044 </td><td> Tested Tet-ücretsiz </td></tr><tr><td> Blasticidin </td><td> Invivogen </td><td> ant-bl-1 </td><td></td></tr><tr><td> Zeocin </td><td> Invivogen </td><td> ant-zn-5 </td><td></td></tr><tr><td> G418 </td><td> PAA laboratuvarları </td><td> P31-011 </td><td> Ortam içinde 100 mg / ml </td></tr><tr><td> Anti-TET02 </td><td> Mobitec GmbH </td><td> TET02 </td><td> WB için 1/1000 ile 1/2000 kullanın </td></tr><tr><td> pcDNA6/TR </td><td> Invitrogen </td><td> V1025-20 </td><td></td></tr><tr><td> pT-Rex DEST30 </td><td> Invitrogen </td><td> 12301-016 </td><td></td&gt; </tr><tr><td> Tetrasiklin </td><td> Sigma </td><td> 87128 </td><td> 2 mg / ml etanol içinde </td></tr><tr><td> Doksisiklin </td><td> Sigma </td><td> D9891 </td><td> 2 mg / ml su içinde </td></tr><tr><td> Klonlama silindirler </td><td> Bellco Glass A.Ş. </td><td> 2090-00808 </td><td> yeniden kullanılabilir </td></tr></tbody></table>

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

Memeli hücre biyolojisi alanında önemli bir yaklaşımı gösterir etmek amacı ile, seçilen hücre hatları ilgi genlerinin ifadesi manipülasyon bir ya da bu genin geçici / kararlı ekspresyonu ya da devirme kullanılarak gen fonksiyonu (ler) in çeşitli ilgi. Ne yazık ki, çeşitli hücre biyolojik araştırmalar için bu yaklaşımın uygun olduğunda hücre büyümesi / çoğalması kusurları veya istenmeyen hücre farklılaşmasında gen ekspresyonu sonucu manipülasyonlar. Bu nedenle, araştırmacılar E. alınan Tetrasiklin repressör proteini (Tetr), adapte olması coli, tetrasiklin direnci operon 1, 2,3, memeli hücrelerinde cDNA ifade etmek için çok verimli ve sıkı düzenleyici sistemleri üretmek için. Kısacası, Tetr de TO tetrasiklin (Tet-off sistemi olarak adlandırılır) ilavesinden sonra yükseltici bölgesinde Tet-operatör (TO) bağlanarak veya (2) bağlama ile transkripsiyon (1) blok başlatma ya da için modifiye edilmiştir ttetrasiklin onun yokluğunda (sistem Tet-on adlandırılır) (Şekil 1). Tet-off sistemi tetrasiklin sürekli varlığı (hangi doku hücre kültürü ortamında yaklaşık 24 saat bir yarılanma ömrüne sahiptir) gerektirir rahatsızlık Verilen Tet-sistem daha kapsamlı çok sıkı geliştirme sonuçlanan optimize edilmiştir ve cDNA ifade vektörü için etkili sistemler burada kullanılan şekliyle. Yakında kısa saç tokası RNA&#39;lar (shRNAs) eksprese 4 memeli hücrelerinde, vektörler gen demonte için RNA interferans (RNAi) kurulması sonrası siRNA&#39;lar 5-11 çok benzer işlevi tanımlanmıştır. Gen hedefleri hücresel hayatta kalmak için gerekli olan zaman stabil tükenmesi mümkün değildir Bununla beraber, bu shRNA aracılı knockdown yaklaşımları, konvansiyonel nakavt stratejiler aynı sınırlama var. Bu sınırlandırmayı aşmak için, van de Wetering ark. 12. shRNA ekspresyon vektörü pSUPER modifiye 5 </suOnları kendi ilgi hedef genlerin tetrasiklin indüklenebilir tükenmesi ile stabil hücre hatları oluşturmak için etkin promotor bölgede TO ekleyerek p&gt;. Burada, biz verimli üretmek için bir yöntem tarif istikrarlı insan Tet-on güvenilir indüklenebilir aşırı veya ilgi gen tükenmesi ya götürmek hücre hatları. Bu yöntemi kullanarak, başarılı yarattı Tet-önemli ölçüde sentrozom 13,14 ve 15,16 sinyalizasyon apoptoz MST / hMOB / NDR çağlayan analizi kolaylaştırdı hücre hatları. Bu yazıda, tercih edilen bizim vektörleri tarif verimli üretilmesi için gerekli olan iki ardışık işleme adımları tanımlayan ek olarak insan Tet-on hücre hatları (Şekil 2). Dahası, nesil için bir protokol taslağını yanısıra insan Tet-on hücre hatları, biz kritik teknik prosedürleri ile ilgili yönlerini ve karakterizasyonu hücreleri Tet-üzerinde görüşecek.

Stabil bir şekilde eksprese eden Tetr RPE-1 hücre hatları ilk karakterizasyonu için bir örneği Şekil 4&#39;te gösterilmektedir. Ebeveyn hücre hattı (negatif kontrol olarak görev yapar) eksojen Tetr proteini ifade etmez ise tüm RPE-1 klonları (Şekil 4, şeritli 1), (şerit 2 ve 5 karşılaştırın) Tetr çeşitli düzeylerde ifade unutmayın. RPE-1 arasında Tetr ekspresyonu Bu varyasyon Tet-on hücre klonlarının bekleniyor, plazmid ifade Tetr ifadesi yana plazmid entegr...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Indüklenebilir ve ilgilenilen genin reversibl cDNA aşırı veya shRNA aracılı knock-down ile insan hücre hatları oluşturmak için bir hızlı ve kolay yolu. Bu yöntem geçici transfeksiyon yöntemleri veya konvansiyonel knockdown / nakavtla stratejileri değiştirmek zordur güvenilir ve son derece tekrarlanabilir işlemek hücre hatları için araştırmacılar sağlar.

Tetrasiklin-indüklenebilir (Tet-on) shRNA veya cDNA Expression Durağan İnsan Hücre Üretimi

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

generation-stable-human-cell-lines-with-tetracycline-inducible-tet-on

Research

JoVE Journal

Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

35.0K Görüntüleme.  UCL Cancer Institute. Indüklenebilir ve ilgilenilen genin reversibl cDNA aşırı veya shRNA aracılı knock-down ile insan hücre hatları oluşturmak için bir hızlı ve kolay yolu. Bu yöntem geçici transfeksiyon yöntemleri veya konvansiyonel knockdown / nakavtla stratejileri değiştirmek zordur güvenilir ve son derece tekrarlanabilir işlemek hücre hatları için araştırmacılar sağlar.

Video: Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

Tripsin ile Pasajlanması tonlar İnsan Embriyonik Kök Hücre hatları

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Biyoloji

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Mikroenjeksiyon Kararlı Transgenik C. elegans Üretimi

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Gen İfadesi Analizi Embriyonik Zebra balığı ve cDNA Sentezi RNA İzolasyon

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

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

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

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

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Biyogüvenlik Geliştirilmiş Polimer complexed Ecotropic lentivirüs İnsan Hücreleri, İletimi

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

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.