Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice.

摘要

Hypermutation of immunoglobulin genes is a key process in antibody diversification. Availability of rapid and easy assays for monitoring Ig hypermutation would aid development of culture systems for hypermutating B cells, as well as screening for individuals deficient in the process. Two such assays are described. The first exploits the non-randomness of hypermutation. The existence of a mutational hotspot in the Ser-31 codon of a transgenic IgV gene allowed use of the polymerse chain reaction (PCR) to detect transgene hypermutation and identify cell populations in which this mutation had occurred. For animals that do not carry Ig transgenes, the hypermutation extends into the region flanking the 3'-side of the rearranged J segments. PCR amplification of the 3'-flank of VDJH rearrangements that involve members of the abundantly-used VHJ558 family provides a large database of mutations where the germ-line counterpart is unequivocally known. This assay was particularly useful for analysing endogenous Ig gene hypermutation in several mouse strains. As a rapid assay for monitoring mutation in the JH flanking region, following denaturation/renaturation, the PCR-amplified JH flanking region DNA from germinal centre B cells yields mismatched heteroduplexes. These can be quantified in a filter binding assay using the bacterial mismatch repair protein MutS [Wagner et al., Nucleic Acids Resrearch (1995) 23, 3944-3948]. Using this assay, antibody hypermutation was shown to proceed in the absence of the p53 tumour suppressor gene product.