Isotype switching by a microinjected mu immunoglobulin heavy chain gene in transgenic mice.

Abstract

Immunization of transgenic mice carrying an immunoglobulin ,s heavy chain resulted in a response dominated by expression of the transgene variable region. Unexpectedly, in a large proportion of the antibody produced by immunized mice, the transgene MATERIALS AND METHODSAnimals. Transgenic C57BL/6 mice were prepared as described (13).DNA Hybridizations. DNAs digested with restriction enzymes were separated by electrophoresis through a 0.8% agarose gel, transferred to nitrocellulose, and hybridized with 32P-labeled fragments (14).Quantitation of Antibodies. Anti-Ar antibodies were quantitated as described (15); wells were developed with 125I-labeled affinity-purified rabbit anti-IgM or anti-IgG. Reactivity with the anti-idiotypic rat mAb AD8 (16) was quantitated by a solid-phase radioimmunoassay similar to that used for anti-Ar, except that wells were coated with mAb AD8 and the labeled developing reagent was preadsorbed with rat immunoglobulin. CRIA was measured as described (17). The R16.7 private idiotype was measured by the method used for CRIA; rabbit anti-R16.7 antiserum was rendered specific for R16.7 by adsorption with normal A/J immunoglobulin and with another CRIA-positive mAb, CB9. mAb CB9 appears to express the germ-line VH36-65 sequence that controls CRIA (P.F.R. and A.N., unpublished results) whereas the VH of R16.7 expresses several somatic mutations (see below). This adsorbed anti-idiotype antiserum reacts with R16.7 but not with nonspecific IgG or any other CRIA+ mAb tested.Sequence Analysis of mRNA.t The primer-extension dideoxynucleotide sequencing procedure was used (18,19 Fig. 1A) by joining a Abbreviations: Ar, p-azophenylarsonate; CRIA, cross-reactive idiotype of anti-Ar antibodies from A/J

Sequence Transfers between Variable Regions in a Mouse Antibody Transgene Can Occur by Gene Conversion

“…1) was used to produce another transgenic line (VV29). VV29 mice immunized with Ars-KLH exhibited serum levels of transgenederived anti-Ars IgG (data not shown) that were similar to previously analyzed VVC transgenic mice (25,30,31). Thus, the FIGURE 1.…”

“…Hybridomas were initially screened for production of anti-Ars IgG using an ELISA that has previously been described (30). Anti-Ars IgG-producing hybrids were then screened for IgG reactive with the AD8 anti-idiotype again using a previously described ELISA (30). Previous studies have shown that many hybridomas from immunized VVC transgenic mice that exhibit transgene sequence transfers lack AD8 reactivity (25).…”

“…Immunized transgenic mice exhibiting sequence transfers as indicated by the RT-PCR/Southern blot assay were then used to produce hybridomas. Hybridomas were initially screened for production of anti-Ars IgG using an ELISA that has previously been described (30). Anti-Ars IgG-producing hybrids were then screened for IgG reactive with the AD8 anti-idiotype again using a previously described ELISA (30).…”

Gene Conversion-Like Sequence Transfers Between Transgenic Antibody V Genes Are Independent of RAD54

“…Transchromosomal isotype switching has been reported for a number of H chain transgenes (7,8,10,(33)(34)(35)(36); however, the mechanisms involved in interchromosomal switching are not clear. Chromosomal translocations have been associated with transgene switching, and some transgene switch sites show features similar to normal class switch DNA recombinations (9).…”

Anomalous rearrangements of the immunoglobulin heavy chain genes in human leukemias support the loop-out mechanism of class switch.

“…However, in mice carrying a ,u-heavy chain transgene it has been shown that class switch could take place even when the transgene was unlinked to the IgH locus ( 14). These workers concluded that, at least in this artificial system, class switching could occur between different chromosomes and thus presumably between (Fig.…”