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 सहायता मैं Thy1.1 और pKat2 वैक्टर महानिदेशक Schatz और pVSV जी, pRSV - रेव से एक तरह का उपहार थे, और pMDLg / pRRE वैक्टर बीआर कलन से एक तरह का उपहार थे.

1. तैयारी के नमूने और कोशिकाओं <ol><li> Overexpression या shRNA constructs और रेट्रोवायरल पैकेजिंग वेक्टर pKat2 या lentiviral पैकेजिंग pVSV जी pMDLg / pRRE, और 25 pRSV - रेव वैक्टर युक्त. डीएनए (midiprep) के एक मध्यम पैमाने पर तैयारी करें 6 μg डीएनए प्रत्येक ?...

<ol>
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Biol. 18, 75-79 (2011).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+exosome+targets+the+AID+cytidine+deaminase+to+both+strands+of+transcribed+duplex+DNA+substrates.">The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates.</a> Cell. 144, 353-363 (2011).</li><li>Yabuki, M., Fujii, M. M., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MRE11-RAD50-NBS1+complex+accelerates+somatic+hypermutation+and+gene+conversion+of+immunoglobulin+variable+regions.">The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.</a> Nat. Immunol. 6, 730-736 (2005).</li><li>Sale, J. E., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TdT-accessible+breaks+are+scattered+over+the+immunoglobulin+V+domain+in+a+constitutively+hypermutating+B+cell+line.">TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line.</a> Immunity. 9, 859-869 (1998).</li><li>Cumbers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+iterative+affinity+maturation+of+antibodies+in+vitro+using+hypermutating+B-cell+lines.">Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines.</a> Nat. Biotechnol. 20, 1129-1134 (2002).</li><li>Papavasiliou, F. N., Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle-regulated+DNA+double-stranded+breaks+in+somatic+hypermutation+of+immunoglobulin+genes.">Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes.</a> Nature. 408, 216-221 (2000).</li><li>Parsa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+mutates+a+non-immunoglobulin+transgene+independent+of+chromosomal+position.">AID mutates a non-immunoglobulin transgene independent of chromosomal position.</a> Mol. Immunol. 44, 567-575 (2007).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+instability+of+V+region+hypermutation+in+the+Ramos+Burkitt's+lymphoma+cell+line.">Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.</a> Int. Immunol. 13, 1175-1184 (2001).</li><li>Ukai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a:T+mutations+is+dependent+on+cellular+environment+but+independent+of+mutation+frequency+and+target+gene+location.">Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.</a> J. Immunol. 181, 7835-7842 (2008).</li><li>Dull, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+class+switching+of+an+IgM++B+lymphoma+clone+CH12F3+to+IgA++cells.">High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells.</a> Int. Immunol. 8, 193-201 (1996).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+expression+of+activation-induced+cytidine+deaminase+(AID),+a+novel+member+of+the+RNA-editing+deaminase+family+in+germinal+center+B+cells.">Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.</a> J. Biol. Chem. 274, 18470-18476 (1999).</li></ol>

जैसा कि पहले चर्चा की, सेल एंटीबॉडी विविधीकरण के लिए लाइन मॉडल एक लोकप्रिय प्रारंभिक बिंदु उपन्यास प्रोटीन है कि एंटीबॉडी विविधीकरण के दौरान विभिन्न चरणों के प्रभाव की पहचान बन गए हैं. हम यहाँ या तो दस...

सुझाव अभिकर्मकों - इनमें से अधिकांश अन्य विक्रेताओं से इसी तरह के उत्पादों के साथ प्रतिस्थापित किया जा सकता है. <table border="1" ><tbody><tr><th> अभिकर्मक के नाम </th><th > कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियाँ </th></tr><tr><td > 6-अच्छी तरह से स्पष्ट टीसी इलाज प्लेटें </td><td > कोर्निंग </td><td> 3516 </td><td ></td></tr><tr><td > 10 एमएल Luer - Loksyringes बी.डी. </td><td > बी.डी. चिकित्सा </td><td > 309604 </td><td ></td></tr><tr><td> 24 अच्छी तरह से स्पष्ट टीसी इलाज प्लेटें </td><td > कोर्निंग </td><td > 3526 </td><td></td></tr><tr><td > 96 अच्छी तरह से स्पष्ट फ्लैट नीचे polystyrene टीसी इलाज microplates </td><td > कोर्निंग </td><td > 3596 </td><td ></td></tr><tr><td > 100 मिमी टीसी इलाज संस्कृति व्यंजन </td><td > कोर्निंग </td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc सिरिंज फिल्टर, 0.45 सुक्ष्ममापी </td><td > पाल लाइफ साइंसेज </td><td > 4604 </td><td ></td></tr><tr><td > अगर </td><td > Teknova </td><td > A7777 </tघ&gt; <td ></td></tr><tr><td > Agarose </td><td > GeneMate </td><td> ई-3120-500 </td><td ></td></tr><tr><td > एम्पीसिलीन </td><td> सिग्मा </td><td > A0166 </td><td> 100 मिलीग्राम / एमएल पानी में </td></tr><tr><td > बी.डी. FACSCanto द्वितीय प्रवाह कोशिकामापी </td><td> बी.डी. बायोसाइंसेज </td><td ></td><td > या इसी तरह </td></tr><tr><td> बी.डी. फाल्कन दौर नीचे polystyrene ट्यूबों </td><td> बी.डी. बायोसाइंसेज </td><td > 352054 </td><td > FACS के लिए </td></tr><tr><td > BOSC 23 कोशिकाओं </td><td > ATCC </td><td > CRL - 11,270 </td><td ></td></tr><tr><td > सीओ 2 37 के सक्षम इनक्यूबेटर ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (Dulbecco संशोधित ईगल मध्यम) </td><td > सिग्मा </td><td> D6429 </td><td ></td></tr><tr><td > FBS (भ्रूण गोजातीय सीरम) </td><td > मिथुन जैव - उत्पाद </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α चूहे CD90.1 एंटीबॉडी CD90/mouse </td><td > BioLegend </td><td > 202503 </td><td > FITCα-Thy1.1 </td></tr><tr><td > FuGENE 6 अभिकर्मक अभिकर्मक </td><td > रॉश </td><td > 11814443001 </td><td></td></tr><tr><td > HEPES बफर समाधान </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi डीएनए पोलीमरेज़ </td><td > KAPA Biosystems </td><td > KK2101 </td><td ></td></tr><tr><td > पौंड (lysogeny शोरबा - Luria) शोरबा पाउडर </td><td > Difco </td><td > 240230 </td><td ></td></tr><tr><td > मिशन टीआरसी shRNA बैक्टीरियल ग्लिसरॉल शेयर </td><td > सिग्मा </td><td ></td><td> shRNA वैक्टर </td></tr><tr><td > NCS (नवजात बछड़ा सीरम) </td><td > मिथुन जैव - उत्पाद </td><td > 100-504 </td><td ></td></tr><tr><td> पीबीएस (फॉस्फेट बफर समाधान) </td><td > Invitrogen </td><td> 70011 </td><td > पानी में 1x को पतला </td></tr><tr><td > पीई α मानव आईजीएम एंटीबॉडी </td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS (पेनिसिलिन स्ट्रेप्टोमाइसिन-glutamine समाधान) </td><td > जैव - पी मिथुनroducts </td><td > 400-110 </td><td ></td></tr><tr><td> Polybrene (hexadimethrine ब्रोमाइड) </td><td > सिग्मा </td><td > 107689 </td><td > 10 मिलीग्राम / एमएल पानी में </td></tr><tr><td > PureYield प्लाज्मिड Midiprep सिस्टम </td><td > Promega </td><td > A2495 </td><td></td></tr><tr><td> Puromycin </td><td> सिग्मा </td><td > P8833 </td><td > 250 μg / एमएल पानी में </td></tr><tr><td > QIAquick जेल निकासी किट </td><td > QIAGEN </td><td > 28706 </td><td ></td></tr><tr><td> रामोस कोशिकाओं (1 आरए) </td><td > ATCC </td><td > CRL - 1596 </td><td ></td></tr><tr><td > RPMI-1640 मध्यम </td><td > सिग्मा </td><td > R8758 </td><td ></td></tr><tr><td > सुपरस्क्रिप्ट द्वितीय </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR फास्ट qPCR किट </td><td > KAPA Biosystems </td><td > KK4601 </td><td ></td></tr><tr><td > Taq पोलीमरेज़ डीएनए </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > Topo टीए क्लोनिंग किट </td><td > Invitroजनरल </td><td > K4520-01 </td><td ></td></tr><tr><td > TRIzol </td><td > Invitrogen </td><td > 15596-026 </td><td ></td></tr><tr><td > जादूगर एसवी जीनोमिक डीएनए शोधन प्रणाली </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > एक्स - लड़की [5 ब्रोमो - 4 क्लोरोफ्लूरोकार्बन 3 indoyl-β डी galatopyranoside] </td><td > Growcells </td><td > सी 5687 </td><td > 40 मिलीग्राम / एमएल DMSO में </td></tr></tbody></table>

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

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 immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

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

हम वर्णन कैसे प्रदर्शन करने के लिए overexpression या shRNA मानव रामोस बी कोशिका लाइन में और कैसे इन कोशिकाओं में दैहिक hypermutation उपाय constructs युक्त. रेट्रोवायरल या lentiviral संक्रमण

रामोस बी कक्ष में या विशिष्ट जीन की Overexpression नॉकडाउन के बाद दैहिक Hypermutation मूल्यांकन

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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Immunology and Infection

Murine Model of CD40-activation of B cells

बी कोशिकाओं की CD40 सक्रियण के murine मॉडल

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

प्रेरण और murine बी कक्ष शुद्ध में कक्षा स्विच पुनर्संयोजन के मूल्यांकन

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Multivirus विशेष टी कोशिकाओं की पीढ़ी को रोकें / allogeneic hematopoietic स्टेम सेल प्रत्यारोपण के बाद वायरल संक्रमण का इलाज

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

सेल भाग्य का पालन<em&gt; ई. कोलाई</em&gt; फेज Lambda द्वारा संक्रमण के बाद

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Detection of True IgE-expressing Mouse B Lineage Cells

सच का पता लगाने के लिए माउस बी वंश प्रकोष्ठों आईजीई व्यक्त

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

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

का पता लगाने और दुर्लभ के संवर्धन विशिष्ट प्रतिजन phenotype और समारोह के विश्लेषण के लिए बी कोशिकाओं

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

ओवेरेक्स्पेशन एंड हेरिंग ऑफ ह्यूमन<em&gt; सीआईएस</em&gt; -प्रेनिलट्रांसफ़्रेज़ इन<em&gt; एस्चेरिचिया कोली</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

दुर्लभ प्रतिजन-विशिष्ट बी कोशिकाओं से रक्त में परिचालित में भेदभावपूर्ण मानव मोनोक्लोनल एंटीबॉडी की पीढ़ी

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

सेलुलर तनाव का आकलन और असतत ऑक्सीटोसिन में सूजन-स्रावी मस्तिष्क नाभिक नवजात चूहे में पहले और बाद में पहली कोलोस्ट्रम खिला

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

Murine सीमांत क्षेत्र बी कोशिकाओं के कतरनी प्रवाह प्रेरित प्रवासन का विश्लेषण इन विट्रो में

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, ...