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

Abstract

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 antibodies 1,2 . 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 recombination 1-3 .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 η) 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 development [11][12][13][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 genes [15][16][17][18] .Ramos -a Burkitt lymphoma cell line that constitutively undergoes SHM -has been a popular cell-line model to study SHM [18][19][20][21][22][23][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). Video LinkThe video component of this article can be found at http://www.jove.com/video/3573/ Protocol 1. Preparing samples and cells 1. 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-Rev 25 . Use 6 μg DNA for each construct and ...

Expression of Constitutive Fusion of Ubiquitin to PCNA Restores the Level of Immunoglobulin A/T Mutations During Somatic Hypermutation in the Ramos Cell Line

“…We report the establishment of a cell line that can be used to address in vitro the mechanism of A/T mutations during the process of somatic hypermutation of Ig genes. The Ramos Burkitt cell line has been widely used to study the mechanism of SHM in vitro (19,37,38). Ramos cells exhibit most of the features of SHM in vivo except that the spectrum of mutations displays a deficiency in A/T mutations.…”

Transcription factor binding at Ig enhancers is linked to somatic hypermutation targeting

“…To determine the functional effects on SHM, we subjected the parental A23 cell line and the KO subclones to an IgM loss assay in which loss of IgM expression, detected by flow cytometry, serves a read‐out for levels of SHM at the Ig loci . We first confirmed that IgM loss was AID‐dependent by assaying AID KO cells (Supporting Information Fig.…”

“…The transfection media was replaced with 2 mL of UltraCULTURE media (Lonza 12–725F) supplemented with 1× Glutamax (Gibco 35050061) and 0.1 mg/mL Normocin 4 h later. Forty‐eight hours after transfection the viral supernatant was collected and Ramos cells were transduced as reported previously .…”

Targeted reduction of cholesterol uptake in cholesterol-addicted lymphoma cells blocks turnover of oxidized lipids to cause ferroptosis

“…Ramos cells were plated at a density of 2.5 × 10 5 cells/ml and infected with the GPX4 lentivirus or empty construct by centrifugation at 3000× rpm for 90 min at room temperature (25 °C), as described previously ( 54 ). The cells were then cultured for 48 h before antibiotic selection was initiated.…”