title: High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions.
abstract: Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.
authors.affiliation: Biotechnology Research Institute, National Research Council, Canada, Montréal, Québec.
authors.forename: M D
authors.initials: MD
authors.lastname: Baker
mesh_terms: D000818:Animals; D002460:Cell Line; D002875:Chromosomes / Q000502:physiology*; D005803:Genes, Immunoglobulin; D006825:Hybridomas / Q000276:immunology; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007075:Immunoglobulin M / Q000032:analysis; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping
chemical_list: D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007148:Immunoglobulin mu-Chains