Point mutation of bacterial artificial chromosomes by ET recombination

Bacterial artificial chromosomes (BACs) offer many advantages for functional studies of large eukaryotic genes. To utilize the potential applications of BACs optimally, new approaches that allow rapid and precise engineering of these large molecules are required. Here, we describe a simple and flexible two-step approach based on ET recombination, which permits point mutations to be introduced into BACs without leaving any other residual change in the recombinant product. Introduction of other modifications, such as small insertions or deletions, is equally feasible. The use of ET recombination to achieve site-directed mutagenesis opens access to a powerful use of BACs and is extensible to DNA molecules of any size in Escherichia coli, including the E. coli chromosome

ET recombination : DNA engineering using homologous recombination in E. coli

Recombinogenic engineering, or the modification and cloning of DNA molecules via homologous recombination, has opened up a new era. In contrast to conventional cloning strategies, recombinogenic engineering can be carried out at virtually any chosen position on a given target DNA molecule (which can be small or large). Any type of modification, including sequence deletions, substitutions, and insertions can be carried out via homologous recombination. Because homologous recombination is a precise process of high fidelity, recombinogenic engineering generates DNA clones efficiently and with high precision

BAC engineering for the generation of ES cell-targeting constructs and mouse transgenes

Bacterial artificial chromosomes (BACs) have become a central tool in functional genomics. This is due to their average cloning capacity (around 150 Kb), which can accommodate most eukaryotic genes along with their full set of regulatory elements, and to their greater convenience in handling over other large cloning vectors like P1-based artificial chromosomes (PACs) and yeast artificial chromosomes (YACs). Two key advances for harnessing the full power of BACs have been the development of methods to modify them with precision and ease (see below), and the ability to use them for transgenesis in higher model organisms, like mouse and zebrafish (for review, see (1))