An element in the endogenous IgH locus stimulates gene targeting in hybridoma cells.
Gene targeting of the immunoglobulin (Ig) heavy chain locus is the basis of improved methods of investigating gene expression and of antibody engineering. The VH-Cmu intron is a convenient region for mediating homologous recombination events which result in production of Ig bearing an altered heavy chain. Also, this segment includes several elements which are important for gene expression, replication and isotype switching: in some cases it will be advantageous to alter these processes by modifying this intron. Considering that multiple targeting steps might be needed to accomplish all the requisite changes, it is important to know whether any of the anticipated modifications also alter the recombinogenicity of the IgH locus. To test this possibility we have measured the frequency at which a mutation in the Cmu3 exon of the endogenous mu gene is corrected by homologous recombination with a transfected segment of Cmu DNA. Comparison of recombination frequencies in several engineered hybridomas indicates that deletion of a 7.1 kb segment from the VH-Cmu intron depresses recombination by approximately 10-fold.
Buzina
A
A
Department of Immunology, University of Toronto, Canada.
Shulman
M J
MJ
D001483:Base Sequence; D017931:DNA Primers; D017353:Gene Deletion; D018390:Gene Targeting; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D007438:Introns; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D014162:Transfection
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007148:Immunoglobulin mu-Chains