Introduction to the Workshop
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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Construction of improved vectors for the preparation of PAC libraries
Eirik Frengen, Panayotis A. Ioannou[1,2], Chira Chen, Eugenia Pietrzak, Xiaoping Guan, Joe Capehart, Joel Jessee, Hans Lehrach and Pieter J. de Jong
Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, BRL/Life technologies, Gaithersburg, MD, Imperial Cancer Research Fund, London, UK.
Recently, we have developed new procedures for the cloning of large DNA fragments using a bacteriophage P1 derived vector, pCYPAC1 . In view of the large sizes (up to 400 kb clones have been observed) and the single copy mode of maintenance, we have designated our clones as "PACs" for "P1-derived artificial chromosomes". A 3-fold redundant PAC library with average insert size of 120-130 kb has been prepared. The library is currently being characterized and copies of the library have been prepared and distributed to many genome centers to ensure wide-spread access to the library. Our experience with the current library is that chimerism is not a problem associated with this cloning approach, that the clones are very stable as compared to conventional cosmid clones and that the cloning procedures are not particularly recombinogenic.
Some of the anticipated uses of PAC clones include expression studies in mammalian cells and the preparation of transgenic animals for the analysis of genes carried on the PAC clone. To facilitate these studies, we are currently improving the PAC-vector as follows. The EBV oriP replication sequences are inserted to ensure stable episomal propagation when a PAC clone is transfected into a human cell for analysis of genes carried on the clone. An eukaryotic dominant-selectable marker gene (Blasticidin) under control of a "universal" promoter (ß-actin promoter) is also inserted in order to enable selection for the desired mammalian clones. These improvements of the vector will be highly useful for expression cloning and selection. The PAC vector will also include sequences important to select for cre/loxP-based site-specific integration in mammalian chromosomes, genome targeting.
A conversion of YACs to PACs would be one strategy to obtain PAC-clones carrying large human genes. We have therefore constructed shuttle vectors that permits the conversion of linear YACs into circular PACs through in vivo homologous recombination procedures in yeast, for subsequent transfer to E.coli. The resulting PACs could then be purified from E. coli and transfected into a mammalian cell for further analysis of cloned genes.
Work was supported in part by a grant from the U.S. Department of Energy (ER61883, P.J. de Jong, P.I.).
 Ioannou, P.A., Amemiya, C.T., Garnes, J., Kroisel, P.M., Shizuya, H., Chen, C., Batzer, M.A. and de Jong, P.J. (1994). A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nature Gen. 6: 84-89.