Homologous recombination in hybridoma cells: dependence on time and fragment length

Mutant hybridoma-myeloma cell lines that are defective in immunoglobulin production are expected to be useful for defining the molecular requirements of immunoglobulin gene expression. The analysis of such mutants would be greatly facilitated if they could be mapped by marker rescue, i.e., by identifying the segments of wild-type DNA that can restore the normal phenotype by homologous recombination with the mutant chromosomal immunoglobulin gene. To assess the feasibility of this type of mapping, we have measured the efficiency with which fragments of wild-type DNA recombine with a mutant hybridoma immunoglobulin gene and restore normal immunoglobulin production. We found that most if not all recombinants were detectable 2 days after DNA transfer and that the frequency of gene restoration increased with increasing length of the transferred mu gene fragments, between 1.2 and 9.5 kilobases. These results indicate that the available technology should be adequate to map mutations in the mu gene to within approximately 1 kilobase.