Name: assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes
Uploaded: 2011-11-01T13:00:00
Description: Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

PMSCV YARDIM-I-Thy1.1 ve pKat2 vektörler DG Schatz ve pVSV-G, pRSV-Rev bir tür hediye ve pMDLg / pRRE vektörler BR Cullen bir tür hediye edildi.

1. Örnekleri ve hücrelerin hazırlanması <ol><li> DNA (midiprep) aşırı ekspresyonu veya shRNA içeren yapıları ve retroviral ambalaj vektör pKat2 veya lentiviral ambalaj vektörler pVSV-G, pMDLg / pRRE ve pRSV-Rev 25 orta ölçekli bir hazırlık gerçekleştirin . Her bir yapı için 6 mg DNA ve ya pKat2 pVSV-g, pMDLg / pRRE her 4 mg (retroviral enfeksiyonları için) veya 1.5 mg DNA ve pRSV-Rev ambalaj vektörler (lentiviral enfeksiyonlar için) kullanın. </li><li> Bosc 23 medya hazırlayın. Tr...

<ol>
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Daha önce tartışıldığı üzere, antikor çeşitlendirilmesi için hücre hattı modelleri, antikor çeşitlendirilmesi sırasında farklı adımlar etkiler roman proteinleri tespit etmek için popüler bir başlangıç ​​noktası haline gelmiştir. Biz burada, Ramos, B-hücre hattı viral enfeksiyon ya knock-aşağı veya eksprese proteinlerine kullanmak için bir yöntem mevcut ve sonra SHM üzerindeki etkisini incelemek. Bu çalışmalar için, biz WT Ramos ve YARDIM hi</sup...

Önerilen reaktifler bunların çoğu diğer satıcılardan benzer ürünleri ile değiştirilebilir. <table border="1" ><tbody><tr><th> Reaktifi Adı </th><th > Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td > 6-net TC ile tedavi edilen levhalar </td><td > Corning </td><td> 3516 </td><td ></td></tr><tr><td > 10 ml BD Luer-Loksyringes </td><td > BD Tıp </td><td > 309604 </td><td ></td></tr><tr><td> 24-iyi net TC-tedavi plakalar </td><td > Corning </td><td > 3526 </td><td></td></tr><tr><td > TC-96-net bir düz dipli polistiren tedavi mikropleytin </td><td > Corning </td><td > 3596 </td><td ></td></tr><tr><td > 100 mm TC-tedavi kültür kaplarına </td><td > Corning </td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc şırınga filtreler, 0.45 mikron </td><td > Pall Yaşam Bilimleri </td><td > 4604 </td><td ></td></tr><tr><td > Agar </td><td > Teknova </td><td > A7777 </td&gt; <td ></td></tr><tr><td > Agaroz </td><td > GeneMate </td><td> E-3120-500 </td><td ></td></tr><tr><td > Ampisilin </td><td> Sigma </td><td > A0166 </td><td> 100 mg / ml suda </td></tr><tr><td > BD FACSCanto II akış sitometresinde </td><td> BD Biosciences </td><td ></td><td > ya da benzer </td></tr><tr><td> BD Falcon yuvarlak alt polistiren tüpler </td><td> BD Biosciences </td><td > 352054 </td><td > FACS için </td></tr><tr><td > Bosc 23 hücreleri </td><td > ATCC </td><td > CRL 11270 </td><td ></td></tr><tr><td > 37 yeteneğine sahip CO 2 inkübatör ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (Dulbecco modifiye Eagle orta) </td><td > Sigma </td><td> D6429 </td><td ></td></tr><tr><td > FBS (fetal sığır serumu) </td><td > İkizler Bio-Ürünler </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α-rat CD90/mouse CD90.1 antikor </td><td > BioLegend </td><td > 202503 </td><td > FITCa-Thy1.1 </td></tr><tr><td > FuGENE 6 Transfeksiyon Reaktif </td><td > Roche </td><td > 11814443001 </td><td></td></tr><tr><td > HEPES tamponu çözümü </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi DNA polimeraz </td><td > KAPA Biyosistem </td><td > KK2101 </td><td ></td></tr><tr><td > LB Broth (lysogeny suyu - Luria) Pudra </td><td > Difco </td><td > 240230 </td><td ></td></tr><tr><td > MİSYON TRC shRNA bakteriyel gliserol stoku </td><td > Sigma </td><td ></td><td> shRNA vektörler </td></tr><tr><td > NCS (yeni doğmuş buzağı serumu) </td><td > İkizler Bio-Ürünler </td><td > 100-504 </td><td ></td></tr><tr><td> PBS (fosfat tampon çözelti) </td><td > Invitrogen </td><td> 70011 </td><td > 1x su içinde seyreltilir </td></tr><tr><td > PE α-insan IgM antikoru </td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS (penisilin-streptomisin-glutamin çözüm) </td><td > İkizler Bio-PÜ r ü nler </td><td > 400-110 </td><td ></td></tr><tr><td> Polybrene (hexadimethrine bromür) </td><td > Sigma </td><td > 107689 </td><td > Suda 10 mg / ml </td></tr><tr><td > PureYield Plazmid Midiprep Sistemi </td><td > Promega </td><td > A2495 </td><td></td></tr><tr><td> Puromycin </td><td> Sigma </td><td > P8833 </td><td > 250 mg / ml suda </td></tr><tr><td > QIAquick jel ekstraksiyon kiti </td><td > QIAGEN </td><td > 28706 </td><td ></td></tr><tr><td> Ramos (RA 1) hücreleri </td><td > ATCC </td><td > CRL-1596 </td><td ></td></tr><tr><td > RPMI-1640 ortamı </td><td > Sigma </td><td > R8758 </td><td ></td></tr><tr><td > Üst Simge II. </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR FAST qPCR kiti </td><td > KAPA Biyosistem </td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA Polimeraz </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > TOPO TA Klonlama kiti </td><td > İnvitrogen </td><td > K4520-01 </td><td ></td></tr><tr><td > TRIzol </td><td > Invitrogen </td><td > 15596-026 </td><td ></td></tr><tr><td > Sihirbazı SV Genomik DNA arıtma sistemi </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > X-Gal [5-bromo-4-chloro-3-indoyl-β-D-galatopyranoside] </td><td > Growcells </td><td > C-5687 </td><td > DMSO 40 mg / ml </td></tr></tbody></table>

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

V (D) J rekombinasyon tarafından oluşturulan düşük afinite antikorları B hücreleri ile onların yaşam başlar. Ancak, bir patojen tespit üzerine, immünglobulin (Ig) geninin değişken (V) bölgesinde yüksek afiniteli antikorlar 1,2, yaklaşık 100.000 kat daha fazla somatik hipermutasyon (SHM) ile genomun geri kalanından daha mutasyona uğramış olan. Buna ek olarak, sınıf anahtarı rekombinasyon (KSS), belirli bir patojen için gerekli olan immün yanıtın türüne bağlı olarak farklı efektör fonksiyonları ile antikor üretir. Hem KSS ve SHM uracils üretmek için DNA&#39;da sitozin kalıntılarının deaminates aktivasyonla indüklenen sitidinin deaminaz (AID), tarafından başlatılan. Bu uracils sonuçta, mutasyon ve 1-3 rekombinasyon, tamir yollarının hata eğilimli formları tarafından işlenir . SHM ve CSR moleküler ayrıntıları mevcut anlayış farelerde çalışmaların bir kombinasyonu gelen, birincil hücreler, hücre hatları ve hücre-free deneyler. Fare modelleri birçok onarım faktörler (örneğin UNG, Msh2, Msh6, Exo1 ve polimeraz η) 4-10 için kritik roller gösteren genetik tırnakları ile altın standart olmaya devam etmektedir. Ancak, bütün genleri nakavt çalışmaları için mükellef bulunmaktadır. Örneğin, birkaç çift iplikli sonu onarım proteinleri tırnakları embryonically ölümcül veya B-hücre gelişimi 11-14 bozar. Ayrıca, bazen SHM veya KSS bir proteinin belirli bir işlevi nakavt neden daha fazla küresel hasar maskelenmiş olabilir. Buna ek olarak, farelerde deneyler bu yana aday genler 15-18 tanımlanması ve karakterize hücre hatları tek tek genlerin ifadesi giderek daha popüler bir ilk adım haline gelmiştir değiştirerek, uzun süreli olabilir . Ramos - yapısal SHM uğrar Burkitt lenfoma hücre hattı - SHM 18-24 incelemek için popüler bir hücre-line model olmuştur. Ramos hücrelerinin bir avantajı yerleşik rahat yarı olmasıSHM, nicel bir ölçüsüdür. Vahşi tip hücreleri mutasyonlar, bazı mutasyonların IgM ifade knock out pick up, IgM ifade ve. Bu nedenle, floresan ile aktive olan hücre tarama (FACS) IgM kaybı assaying SHM düzeyi için hızlı okuma sağlar. SHM daha nicel ölçümü doğrudan antikor genleri sıralama elde edilebilir. Ramos hücreleri transfect zor olduğundan, biz istikrarlı ve türevleri üretmek, sırasıyla aşırı ekspresyonu kaset ya da kısa bir saç tokası RNA (shRNA), ya içeren retroviral veya lentiviral yapıları hücrelerini enfekte artış ya da bireysel bir gen ifade düşürdü. Burada, biz Ramos hücreleri enfekte ve sonra SHM (Şekil 1) belirli genlerin rolünü araştırmak için bu hücrelerin nasıl kullandıklarını açıklar.

Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

Biz aşırı ekspresyonu veya shRNA insan Ramos B-hücre hattı ve bu hücrelerin nasıl somatik hipermutasyon ölçmek için yapıları içeren. Retroviral veya lentiviral enfeksiyonları nasıl gerçekleştirileceği tarif

Spesifik genlerin aşırı ekspresyonu veya demonte sonra Ramos B Hücreleri somatik hipermutasyon Değerlendirilmesi

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

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Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Kanda dolaşan nadir antijen spesifik B hücreleri Discriminative insan monoklonal antikorların üretimi

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Hücresel stres ve ayrık oksitosin salgılayan beyin çekirdekleri içinde yenidoğan sıçan iltihabı önce ve sonra ilk kolostrum besleme değerlendirilmesi

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Yamultma akış kaynaklı geçiş antikorundan marjinal bölge B hücrelerinin in Vitro analizine

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...