Detection of gene targeting by co-conversion of a single nucleotide change during replacement recombination at the immunoglobulin mu heavy chain locus.
A method is described for detecting targeted events at the mu heavy chain gene which relies on co-conversion (or co-exchange) of a point mutation with a selectable marker contained on a replacement vector. The vector, designed for application to IgM producing hybridomas, contains a single nucleotide change within the region of homology with the target gene which encodes a different allotypic determinant of IgM. In a model system where homologous recombination corrected a defective mu gene, the length of homology between this nucleotide change and the position of the double strand break in the vector was found to have a critical influence on the co-conversion frequency. In the vector design ultimately used for targeting in hybridomas, one in 1000-2000 stable transformants produced IgM with the allotype encoded by the exogenous DNA, and Southern blot analysis confirmed that these were derived by targeted integration. The sensitivity of the screening procedure using a monoclonal antibody specific to this allotype enabled a targeted clone to be detected in a pool of stable transformants when present at a frequency at least as low as one per cent. Several different modifications of the target locus were obtained as a consequence of alternative crossover positions and, in some cases, vector DNA concatenation.
Smith
A J
AJ
Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.
Kalogerakis
B
B
D000410:Alanine Transaminase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D005803:Genes, Immunoglobulin; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011863:Radioimmunoassay; D011995:Recombination, Genetic / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D005819:Genetic Markers; D007148:Immunoglobulin mu-Chains; D000410:Alanine Transaminase