INTEGRATION VECTORS FOR ANTIBODY CHIMERIZATION BY HOMOLOGOUS RECOMBINATION IN HYBRIDOMA CELLS

Gene targeting in hybridoma cells provides a tool for generating chime ric antibodies with great ease and at high yield. We present an evalua tion of integration vectors for the chimerization of the immunoglobuli n heavy chain locus which are universally applicable to hybridomas of different isotypes and mouse strains. There are three problems arising with vector integration: (i) the frequent persistence of the parental isotype; (ii) an isotype-dependent aberrant replacement-like recombin ation giving rise to antibodies devoid of the C(H)1 domain; and (iii) secondary recombinations leading to excision of the integrated sequenc e. To overcome these problems, we have systematically evaluated the co nsequences of extending the vector flank. Although the homology length dearly determines the recombination frequency, this effect is counter acted by the secondary recombination, which also correlates to the hom ology length. In contrast, the truncating recombination events are not dependent on the homology length and never lead to re-excision of the construct. To take advantage of the increased genetic stability obtai ned with short flanks, we constructed an enrichment vector which yield s high recombination efficiencies despite using a short flanking seque nce. In addition, irradiation of the cells enhanced homologous recombi nation. The problem of the co-production of two isotypes was overcome by a two-step targeting reaction.