We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunolobulin s heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal p gene. After DNA transfer, we detected G418-resstant transformants that produce normal 1gM. Analysis of the DNA structure of the I gene in these transformants indicates that in four of five cases the p gene has been restored as a result of the integration of a single copy of the transfer vector by a reciprocal homologous recombination event; the fifth case seems to have resulted from gene conversion or double crossover. These results suggest that this technology might be adapted for mapping immungbulin gene mutations by marker rescue and for more convenient engineering of specifically altered inmungbulin.The usual methods for identifying the regulatory elements of the immunoglobulin genes have depended on assaying expression from transferred DNA. Several such elements have been defined by comparing expression from specifically altered immunoglobulin genes (1). A possible weakness of this approach is that the transferred genes are assayed in an abnormal context, either integrated at abnormal chromosomal sites or in an unintegrated form. If a regulatory element were to depend on the normal chromosomal context or to lie outside the gene segment that is assayed, the usual gene transfer methods would be inadequate.In microbial systems, these problems are avoided because the chromosomal genes can be modified by homologous recombination with a segment of transferred DNA. Such a technique would be very useful for introducing precise changes into the chromosomal immunoglobulin genes as well as for mapping regulatory mutations, thus providing an important tool for analyzing immunoglobulin gene expression. To assess the feasibility of this approach, we have tested for homologous recombination between transferred and chromosomal immunoglobulin genes by using, as recipient cells, a mutant hybridoma with a frameshift mutation in the DNA encoding the constant region of the A heavy chain (C,, region) (2). Mutant igm482 cells were harvested by centrifugation and were washed, and 2 x 107 cells were resuspended in 0.45 ml of PBS. Prior to electroporation, the plasmid DNA and igm482 cells were kept on ice. The plasmid DNA and igm482 cells were mixed, subjected to a 700-V, 25-,uF pulse, and placed on ice for 10 min. The cells were then transferred to 40 ml of Dulbecco's modified Eagle's medium (DMEM) containing 13% heatinactivated fetal calf serum and 3.5 x 10 '%o 2-mercaptoethanol (2, 3) and were placed at 370C. Cell viability was determined by trypan blue exclusion after ""12 hr of incubation.IgM Analysis. Plaque-forming cells (PFC) were detected by lysis of 2,4,6-trinitrophenyl (TNP)-coupled erythrocytes by using plaque assa...
