Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

This protocol allows users to study how the immune system produces antibodies to pathogens or commensal microbes. In addition, molecular pathways that regulate immunoglobulin gene diversification can be studied using genetically engineered mice. The protocol uses a combination of common molecular biology techniques including PCR and Sanger sequencing and does not require single cell sorting or deep sequencing.

The Peyer's Patches can be easily confused with the surrounding fat tissue. So visual demonstration of this method may be helpful for those who are unfamiliar with this organ to identify its location and observe its dissection. Begin by laying the mouse on the dissection pad with the abdomen exposed.

Generously spray the body of the mouse with 70%ethanol prior to making any incisions to sterilize the dissection area. Make an incision into the skin across the abdomen and pull simultaneously on both sides of the incision with fingers or forceps. Then cut the peritoneal cavity with scissors to expose the internal organs.

Locate the small intestine and remove it by cutting below the stomach and above the cecum. Remove any connective tissue and fat linking the folds of the small intestine together. Examine the external surface of the small intestine for the Peyer's Patches, which are small oval-shaped structures that appear white below a thin layer of translucent epithelial cells.

Carefully excise all visible Peyer's Patches with scissors and collect them into a 1.5 milliliter micro centrifuge tube containing one milliliter of FACS buffer on ice. If the collected tissue sinks to the bottom of the tube, it is in fact a Peyer's patch and not fat tissue. Place a 40 micro meter filter in a six well dish with one milliliter of cold FACS buffer, then pour the Peyer's Patches from the 1.5 milliliter tube onto the filter.

Use the flat end of the plunger from a one milliliter syringe as a pestle to crush the Peyer's Patches on the filter until only the connective tissue remains. Wash the filter and plunger with one milliliter of cold FACS buffer to release the cells into the six well dish. Collect the cells and filter them through a 40 micrometer strainer cap FACS tube.

Then wash the strainer cap with one milliliter of cold FACS buffer. Pellet the cells by centrifuging them at 600G at four degrees Celsius for five minutes. Then decant the supernatant and resuspend the cells in 0.4 milliliters of cold FACS buffer with FC block.

Incubate samples on ice for 15 minutes then wash and pellet the cells. To stain the Germinal center B cells or GCBCs, resuspend the wild type cells in 80 microliters of FACS buffer. Remove 10 microliters of cells from the wild type Peyer's Patches for each staining control.

Leaving 40 microliters of the cell suspension for the experimental samples. Alternatively, use compensation beads for the staining controls. Stain each of the experimental samples in 500 microliters of cold FACS buffer with 2.5 microliters of PNA Biotin for 15 minutes on ice.

Then wash the PNA stain cells. Stain each sample with 500 microliters of GCBC cocktail in the dark and on ice for 15 minutes, making sure that the cells are fully resuspended in the cocktail. To prepare the single stain controls for the compensation matrix, stain the cells in 500 microliters of cold FACS buffer using the dilution specified in the text.

Incubate the staining controls in the dark on ice for 15 minutes. Add two milliliters of cold FACS buffer to wash all the stain samples and controls. Pellet the cells and discard the supernatant before resuspending the cells in a final volume of 500 microliters of cold FACS buffer to run on the cytometer.

Then, use a cell sorter to collect the B220 positive PNA high cells from each stained experimental sample. To extract the DNA from the Germinal center B cells or GCBCs, resuspend the cells in 500 microliters of DNA extraction buffer and five microliters of Proteinase K.Then precipitate the DNA with 500 microliters of isopropanol and one microliter of glycogen. And mix the tube thoroughly by inverting it five or six times.

After incubating the sample for 10 minutes at room temperature, centrifuge it for 15 minutes at 21, 000G and 25 degrees Celsius. Discard the supernatant and wash DNA pellet with one milliliter of 70%ethanol. Centrifuge the DNA at 21, 000G for 10 minutes, then remove the ethanol and air-dry the pellet for five to 10 minutes.

Resuspend the DNA in 30 microliters of TE buffer and incubate it overnight at 56 degrees Celsius. Perform nested PCR for the JH4 Intron. Normalizing the total amount of genomic DNA used in the first PCR to the least concentrated sample.

After running the PCR products on a gel, excise the amplicon and purify it with a gel extraction kit. Then litigate the amplicon into a plasmid and transform electro competent bacterial cells with two microliters of the ligation reaction. Electroporate the cells at 1.65 kilovolts and rescue them in 600 microliters of SOC media for one hour at 37 degrees Celsius in a shaking incubator at 225 RPM.

Plate 100 microliters of the transformed bacteria onto LB agar plates supplemented with ampicillin and incubate the plates overnight at 37 degrees Celsius. Sequence the plasma DNA from the bacterial colonies and align the sequences obtained for each PCR against the JH4 Intron reference sequence using a Clustal Omega software. Identify differences from the reference sequence as mutations.

The Germinal center B cells were identified by measuring expression of the B220 receptor and binding of Peanut agglutinin. Wild-type Peyer's patches contained an average of 4 million cells per mouse. And approximately 8%of the cells were B220 positive PNA high which is half of that observed in AID knockout mice.

Of the 105 unique sequences obtained from wild type Germinal center B cells, a total of 226 mutations were found. Analysis of the mutation spectrum showed a range of transitions and trans versions at a rate of four times 10 to the negative third mutations per base pair. Only two mutations were identified in 113 AID knockout sequences.

Additionally, each JH4 PCR product from wild type Germinal center B cells, contained one to 25 mutations with multiple mutations frequently found on one sequence. When attempting this protocol, remember to keep the cells on ice to preserve cell viability. After staining the cells, keep the cells in the dark to prevent photobleaching the fluorophores.

To correlate somatic hypermutation with antibody affinity maturation, we recommend using an immunization protocol and assaying for antigen specific antibody titers and V region mutations.