Detection of Immunoglobulin Gene Rearrangements
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Epstein-Barr virus (EBV) has been shown to effect immunoglobulin (Ig) gene rearrangement under certain experimental conditions. When used to establish primordial human B cell clones from bone marrow and liver samples, some clones exhibited abnormal Ig expression (
1
–
5
). B cells from LMP2A transgenic mice can exit the bone marrow and colonize the spleen without surface Ig expression (
6
). These transgenic B cells were less able to appropriately rearrange Ig heavy-chain (HC) genes, but correctly rearranged Ig light-chain (LC) genes. By contrast, latently infected B cells isolated from human peripheral blood have a memory B-cell phenotype, expressing both IgM and IgD (
7
). This chapter details experiments that can be used to monitor Ig gene rearrangements in murine B-cell samples. However, these methods can be modified to monitor human B-cell Ig gene rearrangements as well as T-cell receptor gene rearrangements in lymphoid tissue types by designing specific primers as required. Immunoglobulin (Ig) HC and LC gene rearrangement in murine B cells occurs in a specifically coordinated manner. The genes encoding for the mature polypeptides must be assembled from several gene fragments spaced across several thousand kilobases of genomic DNA. For the Ig HC gene, one of 12 diversity (D
H
) segments joins to one of four joining segments (J
H
) to create a rearranged D-J
H
segment. One of several hundred variable (V
H
) gene segments is then joined to the DJ
H
segment (Fig. 1 ). Once a HC protein is expressed from one allele, rearrangement of the remaining HC locus is allelically excluded (
8
).
Fig. 1.
Schematic of Ig HC gene rearrangements during B-cell development.
