332 COMBINATION OF ZINC FINGER NUCLEASES AND MODIFIED BACTERIAL ARTIFICIAL CHROMOSOMES REVEALS TREMENDOUS RATES OF HOMOLOGOUS RECOMBINATION

Abstract

DNA-based vectors have been used for decades to modify the genomes of mammalian cells by homologous recombination in a specific and site-directed way. Even though various modifications of the procedure have been presented, efficiency is relatively low for many target sites and novel projects still have an unforeseeable outcome. This is in particularly true for site-directed mutagenesis in primary cells intended for use in the generation of large animal models because of their impaired predisposition for homologous recombination compared with stem cells. The recent development of site-specific nucleases is based on a completely different principle: they do not necessarily involve recombination between DNA strands, but rather make use of the inefficient correction of double-strand breaks in the genomic DNA by the cellular DNA repair machinery after such a double-strand break has been introduced by a synthetic enzyme that directed nuclease activity to a defined site in the genome. Here, we intended to evaluate the potential of zinc finger nucleases (ZFN) to introduce a lacZ reporter gene into the CFTR locus. Initially, the efficiency of 3 different ZFN pairs was examined under different conditions revealing modification efficiencies between 0 and 38%. An optimized protocol was used to combine the most efficient ZFN pair with either a bacterial artificial chromosome (BAC) vector or a conventional targeting vector carrying the desired modification. Although the conventional vector failed to introduce the reporter gene in any of more than 200 clones examined, the BAC correctly modified the target site in 32 of 75 clones in a heterozygous way and in 10 out of 75 clones in a homozygous way. However, the introduction of small vector fragments into the CFTR locus in rare cases indicated that the ZFN caused a double-strand break but the vector was not able to act as a recombination donor. On the other hand, transfection of the BAC alone only resulted in 1 modified clone out of 98 and, thus, our data strongly support the hypothesis that the forced introduction of double-strand breaks dramatically increases the rate of homologous recombination, but they also provide indication that the design of the targeting vector has a profound influence on the efficiency.