GENETIC STABILITY OF GENE-TARGETED IMMUNOGLOBULIN LOCI .2. INFLUENCE OF THE CELL-LINE AND THE VECTOR LINEARIZATION SITE

The site-specific integration of exogenous gene fragments by homologou s recombination provides a convenient method for altering the immunogl obulin loci of B cells and specifically designing antibody molecules. To introduce a human isotype into the heavy chain locus of mouse hybri doma cells we compared the recombination frequencies of vectors that c ould be linearized either as integration or as replacement constructs in different cell lines. Integration as well as replacement recombinat ion was observed, irrespective of the location of the site at which th e vector was cleaved. Integration events involving the human IgG1 vect ors were lost at high frequency due to secondary vector excision, so t hat all stable recombinations were found to be replacement events. Rep lacement recombination of an integration vector involves an illegitima te crossover at least at the 3' side and sometimes gives rise to delet ion of the C(H)1 domain. However, a homologous event at the 3' side is more efficient than an illegitimate one, so that a homology that is d istributed on both sides of the heterologous region promotes targeting at higher frequency than a contiguous sequence of the same total leng th. The position of the linearization site in the vector markedly infl uenced the targeting efficiency, but surprisingly, whether a double-st rand break in the homology or in the heterology region more efficientl y promoted integration was dependent on the cell line. In all cells, h owever, cleavage of the vector outside the homology region favoured st able replacements with a bias against C(H)1-truncated clones. We furth er show that the frequency of replacements induced by integration vect ors is not correlated to the homology length and cannot be increased b y irradiation of the cells. Our findings indicate that for targeting t he IgH locus other mechanisms might be involved than at other loci.