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-V-H-D-J(H) and L-V-kappa/lambda-J(kap pa/lambda) regions of mouse immunoglobulin genes. this strategy is bas ed on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the S'-untranslated region and in the intron downstream of the rearranged J(H)/J(kappa/lambda) sequence s. Variable regions with Intact coding sequences, including full-lengt h leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments t hat do not need to be adapted for recombinant antibody expression, thu s facilitating the generation of chimeric and isotype-switched immunog lobulins.