Replacement recombinant events targeted at immunoglobulin heavy chain DNA sequences in mouse myeloma cells.
The rate of gene conversions and double crossovers between transfected and integrated mu heavy chain immunoglobulin genes was measured in myeloma cells. The assay relies on correction of an integrated and defective mu heavy chain expression unit, present in a repeated head to tail array in the genome of the myeloma cell line J558L. Following electroporation of these cells with restriction fragments containing normal immunoglobulin sequences, targeted recombination events are identified by a complement-mediated haemolytic plaque assay measuring production of functional IgM. Recombination results in replacement of a segment of the target sequence with the exogenous sequence. Different crossover positions are possible, giving rise to alternative rearrangements of the target site. In the case of one of the recombinants we analysed, more than one of the repeated targets had undergone a conversion event. The efficiency of homologous recombination was shown to depend on the extent of homology between transfected and target DNA. A targeting efficiency of 1 x 10(-5) to 2 x 10(-5) was achieved when the exogenous DNA contained 10,000 bases of sequence homologous with the target.
Smith
A J
AJ
Medical Research Council, Laboratory of Molecular Biology, Cambridge, England.
Kalogerakis
B
B
D000818:Animals; D002875:Chromosomes / Q000648:ultrastructure; D003434:Crossing Over, Genetic; D004247:DNA / Q000032:analysis*; D005787:Gene Frequency; D006462:Hemolytic Plaque Technique; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007075:Immunoglobulin M / Q000235:genetics; D051379:Mice; D010954:Plasmacytoma / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D004247:DNA