Specific amplification by PCR of rearranged variable regions of immunoglobulin genes from mouse hybridoma cells

Abstract

We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-V(H)-D-J(H) and L-V(κ/λ)-J(κ/λ) 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 J(H)/J(κ/λ) 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.