Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.
We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa/lambda sequences. Variable regions with intact coding sequences, including full-length leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments that do not need to be adapted for recombinant antibody expression, thus facilitating the generation of chimeric and isotype-switched immunoglobulins.
Berdoz
J
J
Swiss Institute for Experimental Cancer Research, University of Lausanne.
Monath
T P
TP
Kraehenbuhl
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D017931:DNA Primers; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006825:Hybridomas / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011544:Pseudogenes
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D015345:Oligonucleotide Probes