Production of mouse V/human C chimeric kappa genes by homologous recombination in hybridoma cells. Analysis of vector design and recombinant gene expression.
Homologous recombination between transferred and chromosomal Ig genes in mouse hybridoma cells offers a general method of altering the chromosomal Ig genes in predetermined ways. Recombination is infrequent in hybridoma cells, and we have been interested in improving the methods for identifying and recovering the rare recombinants. We have used vectors that are designed to replace the mouse chromosomal C kappa segment with the human equivalent, so that recombinants produce mouse V/human C chimeric kappa-chains. We describe an enhancerless, replacement type vector that can be used with the herpes thymidine kinase counterselection to provide such enrichment that homologous recombinants constitute 15% of the selected G418-resistant, FIAU-resistant cells. We have also measured the level of chimeric kappa gene expression and found surprisingly that (1) it is very variable among transformants with the same recombinant gene structure, (2) there is no systematic difference in the level of production by recombinants that retain or have lost the J-C kappa intron enhancer, and (3) the amount of chimeric kappa mRNA in even the highest producing transformants is much less than the amount of the corresponding mouse kappa mRNA.
Sun
W
W
Department of Immunology, University of Toronto, Ontario, Canada.
Xiong
J
J
Shulman
M J
MJ
D000818:Animals; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins; D011995:Recombination, Genetic
D007127:Immunoglobulin Constant Regions; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins