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

The pMSCV-AID-I-Thy1.1 and pKat2 vectors were a kind gift from D. G. Schatz and the pVSV-G, pRSV-Rev, and pMDLg/pRRE vectors were a kind gift from B. R. Cullen.

1. Preparing samples and cells
<ol>
	<li>Perform a medium scale preparation of DNA (midiprep) of the overexpression or shRNA-containing constructs and the retroviral packaging vector pKat2 or the lentiviral packaging vectors pVSV-G, pMDLg/pRRE, and pRSV-Rev25. Use 6 &mu;g DNA for each construct and either 4 &mu;g of pKat2 (for retroviral infections) or 1.5 &mu;g DNA for each of the pVSV-g, pMDLg/pRRE, and pRSV-Rev packaging vectors (for lentiviral infections).</li>
	<li>Prepare BOSC 23 media. For transfectio...

<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. 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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. 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As discussed previously, cell line models for antibody diversification have become a popular starting point to identify novel proteins that influence different steps during antibody diversification. We present here a method for using viral infection to either knock-down or overexpress proteins in the Ramos B-cell line and then examine the impact on SHM.
For these studies, we utilize both WT Ramos and AIDhi Ramos cells. WT cells can be used to study factors involved in targeting or e...

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<td >6-well clear TC-treated plates</td>
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<td>3516</td>
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<td >10 mL BD Luer-Loksyringes</td>
<td >BD Medical</td>
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<td>24-well clear TC-treated plates</td>
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<td >3526</td>
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<td >96-well clear flat bottom polystyrene TC-treated microplates</td>
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<td >3596</td>
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<td >100 mm TC-treated culture dishes</td>
<td >Corning</td>
<td >430167</td>
<td >&nbsp;</td>
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<td >Acrodisc syringe filters, 0.45 &mu;m</td>
<td >Pall Life Sciences</td>
<td >4604</td>
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<td >Agar</td>
<td >Teknova</td>
<td >A7777</td>
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<td >Agarose</td>
<td >GeneMate</td>
<td>E-3120-500</td>
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<td >Ampicillin</td>
<td>Sigma</td>
<td >A0166</td>
<td>100 mg/mL in water</td>
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<td >BD FACSCanto II flow cytometer</td>
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<td >or similar</td>
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<td>BD Falcon round bottom polystyrene tubes</td>
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<td >352054</td>
<td >for FACS</td>
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<td >BOSC 23 cells</td>
<td >ATCC</td>
<td >CRL-11270</td>
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<td >CO2 incubator capable of 37&deg;C</td>
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<td >DMEM (Dulbecco&prime;s modified Eagle&prime;s medium)</td>
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<td >FBS (fetal bovine serum)</td>
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<td >100-106</td>
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<td >BioLegend</td>
<td >202503</td>
<td >FITC &alpha;-Thy1.1 </td>
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<td >FuGENE 6 Transfection Reagent</td>
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<td >11814443001</td>
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<td >HEPES buffer solution</td>
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<td >KAPA HiFi DNA polymerase</td>
<td >KAPA Biosystems</td>
<td >KK2101</td>
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<td >MISSION TRC shRNA bacterial glycerol stock</td>
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<td >NCS (newborn calf serum)</td>
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<td >100-504</td>
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<td>PBS (phosphate buffered solution)</td>
<td >Invitrogen</td>
<td>70011</td>
<td >diluted to 1x in water</td>
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<td >PE &alpha;-human IgM antibody</td>
<td >BioLegend</td>
<td >314508</td>
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<td >PGS (penicillin-streptomycin-glutamine solution)</td>
<td >Gemini Bio-Products</td>
<td >400-110</td>
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<td>Polybrene (hexadimethrine bromide)</td>
<td >Sigma</td>
<td >107689</td>
<td >10 mg/mL in water</td>
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<td >PureYield Plasmid Midiprep System</td>
<td >Promega</td>
<td >A2495</td>
<td>&nbsp;</td>
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<td>Puromycin</td>
<td>Sigma</td>
<td >P8833</td>
<td >250 &mu;g/mL in water</td>
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<td >QIAquick gel extraction kit</td>
<td >QIAGEN</td>
<td >28706</td>
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<td>Ramos (RA 1) cells</td>
<td >ATCC</td>
<td >CRL-1596</td>
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<tr>
<td >RPMI-1640 medium </td>
<td >Sigma</td>
<td >R8758</td>
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<td >SuperScript II</td>
<td >Invitrogen</td>
<td >18064-022</td>
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<td >SYBR FAST qPCR kit</td>
<td >KAPA Biosystems</td>
<td >KK4601</td>
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<td >Taq DNA Polymerase</td>
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<td >TOPO TA Cloning kit</td>
<td >Invitrogen</td>
<td >K4520-01</td>
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<td >TRIzol</td>
<td >Invitrogen</td>
<td >15596-026</td>
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<td >Wizard SV Genomic DNA purification system</td>
<td >Promega</td>
<td >A2361 </td>
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<td >X-Gal [5-bromo-4-chloro-3-indoyl-&beta;-D-galatopyranoside]</td>
<td >Growcells</td>
<td >C-5687</td>
<td >40 mg/mL in DMSO</td>
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</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

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

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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