Integration vectors for antibody chimerization by homologous recombination in hybridoma cells.
Gene targeting in hybridoma cells provides a tool for generating chimeric antibodies with great ease and at high yield. We present an evaluation of integration vectors for the chimerization of the immunoglobulin 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 recombination giving rise to antibodies devoid of the CH1 domain; and (iii) secondary recombinations leading to excision of the integrated sequence. To overcome these problems, we have systematically evaluated the consequences of extending the vector flank. Although the homology length clearly determines the recombination frequency, this effect is counteracted by the secondary recombination, which also correlates to the homology 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 obtained with short flanks, we constructed an enrichment vector which yields high recombination efficiencies despite using a short flanking sequence. In addition, irradiation of the cells enhanced homologous recombination. The problem of the co-production of two isotypes was overcome by a two-step targeting reaction.
PubMed Abstracts
PubMed Abstracts