Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells

To facilitate the use of large-insert bacterial clones for functional analy sis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein-Barr virus re plicon, oriP, is included to ensure stable episomal propagation of the larg e insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable sel ection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeti ng in mammalian chromosomes are also present. In addition, we demonstrate t hat the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been inclu ded in the vector to enable linearization of the large-insert clones, e.g., for optical mapping studies. The pPAC4 vector has been used to generate li braries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experime nts, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene i nto mutant cells or transgenic or knock-out animals. (C) 2000 Academic Pres s.