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 transfection, use both unamended Dulbecco&prime;s modified Eagle&prime;s medium (DMEM) as well as complete DMEM. To make complete DMEM, add 1% HEPES buffer, 1% penicillin-streptomycin-glutamine (PGS), and 10% newborn calf serum (NCS) to a bottle of DMEM.</li>
	<li>Prepare Ramos media. For the infection step, make complete RPMI-1640 medium (RPMI) by adding 1% HEPES, 1% PGS, and 10% fetal bovine serum (FBS) to a bottle of RPMI. For single cell seeding step, make "conditioned" media by growing Ramos cells in complete RPMI for 2 days, spinning down the cells down at 400 X g for 4 minutes and filtering the supernatant with a 0.2 &mu;m filter into a sterile bottle. Like other complete media, conditioned media is stored at 4&deg;C until needed.</li>
	<li>Split 3 x 106 BOSC 23 cells into a 100-mm plate in 10 mL complete DMEM the day before transfection so that the cells will be at 50-60% confluency the day of transfection. Make one plate of cells for each vector you plan on transfecting. It is important that the cells have not been kept in culture for too long; use low passage number cells or recently thawed cells for both the transfection and infection steps to insure a good infection.</li>
	<li>Include controls for your experiments. For transfections and infections, create a negative control (untransfected or uninfected cells) and a positive control (virus that expresses GFP and puromycin-resistance gene). For shRNA knock-down experiments, infect a well with a scrambled or irrelevant shRNA control.</li>
</ol>
2. Transfecting BOSC 23 cells
<ol>
	<li>Warm the bottles of unamended DMEM and complete DMEM at 37&deg;C.</li>
	<li>Remove media from the BOSC 23 cells and replace with 5 mL of complete DMEM. Return the cells to a 37&deg;C incubator until use in step 2.6.</li>
	<li>Warm the bottle of FuGENE 6 at room temperature for 5 minutes then briefly vortex (&lt;1 second).</li>
	<li>Aliquot 600 &mu;L unamended DMEM into a 1.5 mL microcentrifuge tube.</li>
	<li>Dilute 30 &mu;L FuGENE 6 into the 600 &mu;L unamended DMEM, vortex briefly (&lt;1 second), and then incubate at room temperature for five minutes. Be careful to pipette directly into the media. Do not let the FuGENE 6 touch the sides of the tube.</li>
	<li>Add 6 &mu;g construct and either 4 &mu;g pKat2 (retroviral infections) or 1.5 &mu;g each of pVSV-G, pMDLg/pRRE, and pRSV-Rev (lentiviral infections) to the DMEM/FuGENE 6 mix, vortex briefly (&lt;1 second), and then incubate at room temperature for 25 minutes.</li>
	<li>Add the DNA/DMEM/FuGENE 6 mix to the BOSC 23 cells and return to a 37&deg;C incubator for 24 hours.</li>
	<li>After 24 hours, add 5 mL complete DMEM to the transfected cells and return to a 37&deg;C incubator for another 24 hours.</li>
</ol>
3. Harvesting viral particles
<ol>
	<li>Harvest the 10 mL of media from the transfected BOSC 23 cells with a 10 mL syringe.</li>
	<li>Attach a 0.45 &mu;m filter to the end of the syringe and filter the media into a clean 15 mL polypropylene tube.</li>
	<li>Use the viral media immediately or freeze as 1 mL aliquots in 2 mL cryogenic vials at -80&deg;C.</li>
	<li>After harvesting the viral media, confirm that efficient transfection has occurred in the positive control cells by FACS. This can also be done for all samples if the construct contains either a fluorescent (e.g. GFP) or a cell-surface marker (e.g. Thy1).</li>
</ol>
4. Infecting B cells
<ol>
	<li>Seed 2 mL of 2 x 105 cells/mL Ramos cells in complete RPMI into a 6-well plate 24 hours before infection. Again, be sure to use low passage number cells or recently thawed cells to insure good infection.</li>
	<li>If using frozen aliquots of virus, thaw the viral media quickly at 37&deg;C.</li>
	<li>Mix 1 mL viral media with 3 &mu;L of 10 mg/mL polybrene. The final concentration of polybrene should be 10 &mu;g/mL in a total volume of 3 mL.</li>
	<li>Add the virus/polybrene mix to the cells and mix well by pipetting. As mentioned earlier, one well should be used as an uninfected control; add 1 mL complete RPMI and 3 &mu;L polybrene to this well instead of 1 mL virus. Be sure to infect one well with a matched negative control. We also infect one well with a GFP-containing vector, to determine infection efficiency by FACS.</li>
	<li>Centrifuge the 6-well plate at 3000 X g for 90 minutes at room temperature.</li>
	<li>Return the plate to a 37&deg;C incubator for 48 hours.</li>
	<li>After 48 hours, spin down the infected cells in a 15 mL polypropylene tube at 400 X g for 4 minutes at room temperature. Remove the media by aspiration, being sure to not disturb the cell pellet. Resuspend the cells in fresh RPMI.</li>
	<li>Confirm the efficiency of infection in the positive control sample by FACS. As before, you can do this for all your samples if the construct contains either a fluorescent (e.g. GFP) or a cell-surface marker (e.g. Thy1).</li>
	<li>Start selection of cells (if appropriate). Our shRNA-containing constructs all contain a puromycin resistance cassette. We select shRNA-infected Ramos cells with 0.25 &mu;g/mL puromycin. We find that derivatives of Ramos cells sometimes show altered susceptibility to puromycin and, therefore, we perform kill curves to optimize the concentration of puromycin for each derivative. Also treat uninfected control cells with puromycin to confirm that selection is effective.</li>
</ol>
5. Single cell seeding
Single seeding can be performed in one of two ways: by FACS sorting or manually.
<ol>
	<li>FACS: Use a FACS sorter with a plate adapter to seed 1 cell/well in a 96-well plate pre-filled with 100 &mu;L of 40% RPMI/40% conditioned media/20% FBS. This method gives a lot more single cell clones per plate.</li>
	<li>Manually: Count the cells. Dilute an aliquot of cells to 10 cells/mL in 40% RPMI/40% conditioned media/20% FBS. Use 10 mL of diluted cells for each seeded plate. Different clones will show variable recovery at this stage therefore it is useful to set up several plates at different concentrations (ranging from 3 to 20 cells/mL). Use the one that produces fewer outgrowths to avoid clones arising from multiple cells). Use a multichannel pipette to seed 96-well plates with 100 &mu;L of diluted cells.</li>
	<li>Place the plate in a 37&deg;C incubator for 2 weeks.</li>
	<li>Check individual wells under the microscope to confirm the presence of cells one day after seeding. Single cells can be tricky to find due to the need to focus at the correct depth. Focusing on the well numbers imprinted at the bottom of the 96-well plate should help. Cells are easier to find when seeded by the FACS sorter since most wells have a cell.</li>
	<li>After 2 weeks, growing colonies can be barely seen by naked eye by looking at the plates from below. They are much easier to see under the microscope. Colonies tend to grow along the edges of the well and typically most colonies in a plate grow along the same edge in different wells. Mark wells with robust cell growth and transfer the cells to 24-well plates in 1 mL RPMI and return to a 37&deg;C incubator.</li>
	<li>Maintain cells at 1 x 105 to 1 x 106 cells/mL. Change the media by spinning the cells at 400 X g for 4 minutes at room temperature and removing the media as needed. Replace with 1 mL fresh RPMI.</li>
	<li>After several days of growth, transfer the cells to a 6-well plate and continue growing in a 37&deg;C incubator.</li>
	<li>After 3 weeks, analyze the clones for IgM loss (as described below).</li>
	<li>Continue to grow the cells for 2 more weeks and then analyze the clones again at the 5 week time point.</li>
</ol>
6. Analysis: IgM loss (3 weeks and 5 weeks)
<ol>
	<li>Aliquot 5 x 105 cells from each well into a 1.5 mL microcentrifuge tube and spin the cells down at 400 X g for 4 minutes at room temperature. Use cells from each of the control and shRNA-knock-down wells, as well as one sample for an unstained control.</li>
	<li>Wash the cells in 1 mL of PBS and then spin again at 400 X g for 4 minutes at room temperature.</li>
	<li>Resuspend in 50 &mu;L of PBS (unstained control) or PBS + 1:20 dilution of PE-labeled &alpha;-human IgM antibody. Incubate on ice for 1 hour.</li>
	<li>Spin the cells down at 400 X g for 4 minutes at room temperature.</li>
	<li>Wash the cells twice with 1 mL of PBS and spin for 400 X g for 4 minutes each time at room temperature.</li>
	<li>Remove the supernatant and resuspend in 100 &mu;L of PBS. Transfer the resuspended stained cells to a FACS tube.</li>
	<li>Analyze the cells using a FACS machine.</li>
	<li>Compare the percentage of IgM- cells from the controls vs. the shRNA knocked-down cells. As noted before, IgM loss as measured by FACS is a reliable semi-quantitative measure of SHM in Ramos cells.</li>
</ol>
7. Analysis: Confirm knock-down by quantitative RT-PCR (5 weeks)
<ol>
	<li>Aliquot 3 x 106 cells from each well and spin down at 400 X g for 4 minutes at room temperature.</li>
	<li>Prepare RNA from the aliquot of cells. We use TRIzol according to manufacturer's recommendations.</li>
	<li>Prepare cDNA from the RNA prep. We use 2 &mu;g RNA and Superscript II according to manufacturer's recommendations.</li>
	<li>Perform a quantitative PCR using the cDNA to confirm knock-down. We use GAPDH as a normalization control, and the SYBR FAST qPCR kit in a 10 &mu;L total reaction volume.</li>
</ol>
8. Analysis: Genomic sequencing (5 weeks)
<ol>
	<li>Aliquot 5 x 106 cells from each well and spin down at 400 X g for 4 minutes at room temperature.</li>
	<li>Isolate genomic DNA from these cells. We use the Wizard SV genomic DNA purification kit according to manufacturer's recommendations.</li>
	<li>PCR amplify the genomic DNA for the Ig V genes19 (Table 1) and any other genes of interest using the KAPA HiFi DNA polymerase. We use this polymerase because of its low error-rates, but any high-fidelity PCR polymerase should be acceptable.</li>
	<li>Gel purify the PCR products on a 1% agarose gel using the QIAquick gel extraction kit.</li>
	<li>Some polymerases add a single deoxyadenosine (A) residue to the 3&prime; ends of the PCR products, while others do not. These 3&prime; A tails are necessary for the TOPO TA cloning step. The KAPA HiFi polymerase does not leave 3&prime; A tails. A-tail the PCR products by incubating 25 &mu;L gel-purified PCR product with 3 &mu;L 10x Taq reaction buffer, 1 &mu;L 10 mM dNTPs, and 1 unit Taq polymerase at 72&deg;C for 30 minutes.</li>
	<li>Clone the gel-purified PCR products using the TOPO TA Cloning kit. Transform the products from the TOPO reaction into the provided E. coli DH5&alpha;-T1R competent cells and plate on LB/agar plates containing 100 &mu;g/mL ampicillin and supplemented with 1.6 mg of X-gal according to manufacturer's recommendations.</li>
	<li>Send individual colonies for sequencing.</li>
	<li>Perform sequence analysis to determine types and locations of mutations.</li>
</ol>
9. Representative Results:
As noted before, we use a positive control viral vector that expresses both GFP as well as a puromycin-resistance gene. We typically see 50-75% GFP+ cells two days after transfection. In our infections, we typically see 50-70% cells are GFP+ two days after infection but before selection. After selection is complete, &gt;95% of cells are GFP+.
As representative experiments, we show the effects of overexpressing AID or knocking down a repair factor in Ramos cells. Specifically, we transfected a retroviral overexpression vector for AID linked via an IRES site to Thy1.1 along with the retroviral packaging vector pKat2 into BOSC 23 cells. Virus-containing media was filtered and then used to infect Ramos cells. Both wild type (WT) and AID overexpressing (AIDhi) cells were single cell seeded and grown for 3 weeks, at which point they were analyzed for gene expression by qRT-PCR (Figure 2) and for IgM loss by FACS (Figure 3). For the FACS staining, the cells were incubated with both 1:20 diluted PE-labeled &alpha;-human IgM antibody as well as 1:400 diluted FITC-labeled &alpha;-rat CD90/mouse CD90.1 (Thy1.1) antibody. In a separate experiment, lentiviruses containing either an irrelevant shRNA (control) or an shRNA against a high-fidelity DNA repair factor expected to protect against SHM were made in BOSC 23 cells, and then infected into an AIDhi clone of Ramos cells. Again, gene expression (Figure 4) and IgM loss (Figure 5) were analyzed in single cell clones after 3 weeks. Since the repair factor is thought to provide protection against mutations during SHM, we see an increase in IgM loss and SHM in the absence of the factor. If we had knocked-down a factor involved in generating SHM-associated mutations, we would have seen a decrease in IgM loss instead.
As expected, our qRT-PCR results show a marked increase in AID expression following infection of cells with an AID overexpression vector (Figure 2), while levels of the DNA repair factor drop in the presence of a targeted shRNA against that factor (Figure 4). Because individual clones may vary, you might see some variation in gene expression levels. Because gene expression can vary, it is necessary to confirm the expression in each clone you plan on analyzing further. Different shRNAs against the same gene might have different effects on gene expression as well, so you should screen several shRNAs to determine which has the greatest impact. Knockdown may also be confirmed at the protein level by immunoblotting. Likewise, individual clones might vary greatly in levels of IgM loss. Some clones might demonstrate a "jackpot" effect, where an IgM mutation occurred in one of the earliest cell divisions - for example, you will see &gt;50% IgM loss simply because one of the cells became IgM- at the two-cell stage. The influence of these clones is minimized by calculating the median for several single cell clones.
<img alt="Figure 1" src="/files/ftp_upload/3573/3573fig1.jpg" /> 
Figure 1. Schematic for the transfection, infection and analysis of IgM loss. See text for details.
<img alt="Figure 2" src="/files/ftp_upload/3573/3573fig2.jpg" /> 
Figure 2. AID overexpression in Ramos cells. WT and AIDhi Ramos cells were single cell cloned and grown for 3 weeks, at which point AID expression in individual clones was measured by qRT-PCR. GAPDH expression was used for normalization. WT was set to 1. The median value is indicated. * indicates a p value of &lt;0.01, calculated as a one-tailed Student&prime;s t-test.
<img alt="Figure 3" src="/files/ftp_upload/3573/3573fig3.jpg" /> 
Figure 3. Increased IgM loss in AIDhi cells as compared to WT cells. Surface IgM was measured by FACS at the 3 week time point, and the percentage of IgM loss was calculated for WT and AIDhi cells. (A) Representative FACS plot of a WT clone and an AIDhi clone. The AID overexpression vector has AID linked to Thy1.1 via an IRES site, so Thy1.1 expression is used as a surrogate for AID expression. (B) Quantitation of IgM loss in several WT and AIDhi clones. The median value is indicated. * indicates a p value of &lt;0.01, calculated as a one-tailed Student's t-test.
<img alt="Figure 4" src="/files/ftp_upload/3573/3573fig4.jpg" /> 
Figure 4. Knockdown of a repair factor in AIDhi cells. Cells were infected with either an irrelevant shRNA control (control) or our shRNA of interest (shRNA), and single cell seeded. After 3 weeks, gene expression in individual clones was measured by qRT-PCR. GAPDH expression was used for normalization. WT was set to 1. The median value is indicated. * indicates a p value of &lt;0.01, calculated as a one-tailed Student's t-test.
<img alt="Figure 5" src="/files/ftp_upload/3573/3573fig5.jpg" /> 
Figure 5. Increased IgM loss in AIDhi cells with knocked-down levels of a repair factor. Surface IgM in several clones was measured by FACS at the 3 week time point, and the percentage of IgM loss was calculated in cells still overexpressing AID. The median value is indicated. * indicates a p value of &lt;0.01, calculated as a one-tailed Student&prime;s t-test.
<img src="/files/ftp_upload/3573/3573table1.jpg" alt="Table 1" /> 
Table 1. Primer sequences for the Ig genes19: the constant region C&mu;, the heavy chain V region (VH), and the light chain V region (VL).

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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. 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>

No conflicts of interest declared.

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...

Suggested reagents &#x2013; most of these may be substituted with similar products from other vendors.
<table border="1" >
<tbody>
<tr>
<th>Name of the reagent</th>
<th >Company</th>
<th>Catalogue number</th>
<th>Comments</th>
</tr>
<tr>
<td >6-well clear TC-treated plates</td>
<td >Corning</td>
<td>3516</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >10 mL BD Luer-Loksyringes</td>
<td >BD Medical</td>
<td >309604</td>
<td >&nbsp;</td>
</tr>
<tr>
<td>24-well clear TC-treated plates</td>
<td >Corning</td>
<td >3526</td>
<td>&nbsp;</td>
</tr>
<tr>
<td >96-well clear flat bottom polystyrene TC-treated microplates</td>
<td >Corning</td>
<td >3596</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >100 mm TC-treated culture dishes</td>
<td >Corning</td>
<td >430167</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >Acrodisc syringe filters, 0.45 &mu;m</td>
<td >Pall Life Sciences</td>
<td >4604</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >Agar</td>
<td >Teknova</td>
<td >A7777</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >Agarose</td>
<td >GeneMate</td>
<td>E-3120-500</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >Ampicillin</td>
<td>Sigma</td>
<td >A0166</td>
<td>100 mg/mL in water</td>
</tr>
<tr>
<td >BD FACSCanto II flow cytometer</td>
<td>BD Biosciences</td>
<td >&nbsp;</td>
<td >or similar</td>
</tr>
<tr>
<td>BD Falcon round bottom polystyrene tubes</td>
<td>BD Biosciences</td>
<td >352054</td>
<td >for FACS</td>
</tr>
<tr>
<td >BOSC 23 cells</td>
<td >ATCC</td>
<td >CRL-11270</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >CO2 incubator capable of 37&deg;C</td>
<td >&nbsp;</td>
<td >&nbsp;</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >DMEM (Dulbecco&prime;s modified Eagle&prime;s medium)</td>
<td >Sigma</td>
<td>D6429</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >FBS (fetal bovine serum)</td>
<td >Gemini Bio-Products</td>
<td >100-106</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >FITC &alpha;-rat CD90/mouse CD90.1 antibody </td>
<td >BioLegend</td>
<td >202503</td>
<td >FITC &alpha;-Thy1.1 </td>
</tr>
<tr>
<td >FuGENE 6 Transfection Reagent</td>
<td >Roche</td>
<td >11814443001</td>
<td>&nbsp;</td>
</tr>
<tr>
<td >HEPES buffer solution</td>
<td >Invitrogen</td>
<td >15630-080</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >KAPA HiFi DNA polymerase</td>
<td >KAPA Biosystems</td>
<td >KK2101</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >LB Broth (lysogeny broth - Luria) Powder</td>
<td >Difco</td>
<td >240230</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >MISSION TRC shRNA bacterial glycerol stock</td>
<td >Sigma</td>
<td >&nbsp;</td>
<td>shRNA vectors</td>
</tr>
<tr>
<td >NCS (newborn calf serum)</td>
<td >Gemini Bio-Products</td>
<td >100-504</td>
<td >&nbsp;</td>
</tr>
<tr>
<td>PBS (phosphate buffered solution)</td>
<td >Invitrogen</td>
<td>70011</td>
<td >diluted to 1x in water</td>
</tr>
<tr>
<td >PE &alpha;-human IgM antibody</td>
<td >BioLegend</td>
<td >314508</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >PGS (penicillin-streptomycin-glutamine solution)</td>
<td >Gemini Bio-Products</td>
<td >400-110</td>
<td >&nbsp;</td>
</tr>
<tr>
<td>Polybrene (hexadimethrine bromide)</td>
<td >Sigma</td>
<td >107689</td>
<td >10 mg/mL in water</td>
</tr>
<tr>
<td >PureYield Plasmid Midiprep System</td>
<td >Promega</td>
<td >A2495</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Puromycin</td>
<td>Sigma</td>
<td >P8833</td>
<td >250 &mu;g/mL in water</td>
</tr>
<tr>
<td >QIAquick gel extraction kit</td>
<td >QIAGEN</td>
<td >28706</td>
<td >&nbsp;</td>
</tr>
<tr>
<td>Ramos (RA 1) cells</td>
<td >ATCC</td>
<td >CRL-1596</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >RPMI-1640 medium </td>
<td >Sigma</td>
<td >R8758</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >SuperScript II</td>
<td >Invitrogen</td>
<td >18064-022</td>
<td>&nbsp;</td>
</tr>
<tr>
<td >SYBR FAST qPCR kit</td>
<td >KAPA Biosystems</td>
<td >KK4601</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >Taq DNA Polymerase</td>
<td >Invitrogen</td>
<td >18038-042</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >TOPO TA Cloning kit</td>
<td >Invitrogen</td>
<td >K4520-01</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >TRIzol</td>
<td >Invitrogen</td>
<td >15596-026</td>
<td >&nbsp;</td>
</tr>
<tr>
<td >Wizard SV Genomic DNA purification system</td>
<td >Promega</td>
<td >A2361 </td>
<td >&nbsp;</td>
</tr>
<tr>
<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>
</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

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 ...

assessing-somatic-hypermutation-ramos-b-cells-after-overexpression-or

Research

JoVE Journal

Immunology and Infection

18.1K Views.  Duke University. 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.

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

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

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 immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

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

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

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

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

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

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

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

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

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

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 and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

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

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

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

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

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

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 of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

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

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, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

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, ...