HIGH-EFFICIENCY SITE-SPECIFIC MODIFICATION OF THE CHROMOSOMAL IMMUNOGLOBULIN LOCUS BY GENE TARGETING

Site-specific modification of the chromosomal immunoglobulin locus by gene targeting is a powerful tool in studying the molecular requiremen ts for immunoglobulin gene structure and function and in the productio n of engineered antibodies. Here, we describe a two step- integration then excision-gene targeting procedure for introducing planned genetic alterations into the chromosomal immunoglobulin locus. The efficiency of gene targeting with an enhancer-trap vector in which an enhancerle ss neo and HSV-tk gene were inserted into the vector backbone was comp ared to that of the corresponding enhancer-positive vector. Both inser tion vectors also contained homology to the chromosomal immunoglobulin target locus along with the desired genetic alteration. The first ste p involved insertion of the transferred vector into the target locus b y homologous recombination. An approximately 15-fold enrichment in the frequency of vector insertion was obtained with the enhancer-trap com pared to the enhancer-positive vector. The majority of targeted cells (75%) contained a single copy of the vector integrated into the chromo somal immunoglobulin locus. The second step involved excision of the i ntegrated vector by intrachromosomal homologous recombination between the duplicated region of homology that removed the integrated vector, neo and tk genes along with one copy of homologous DNA. Vector excisio n was very efficient generating G418(S) FIAU(R) secondary recombinants at the high rate of similar to 10(-3)/cell generation. In the seconda ry recombinants, the overall structure of the chromosomal immunoglobul in locus was restored with the desired genetic alteration being presen t in an expected proportion of the cells. (C) 1998 Elsevier Science B. V. All rights reserved.