To complement [some of the problems of] current YAC libraries, we have recently developed new cloning procedures to create large-insert libraries of human and mouse genomes in low-copy number bacterial plasmids. The plasmid vector (pCYPAC) is derived from Sternberg's bacteriophage p1 vector (pAd10SacBII) and maintains all the sophisticated features of the earlier vector. In addition, the vector includes modifications to construct libraries by electroporation instead of in vitro packaging. Electroporation has the advantage that it avoids the size constraints imposed by the PI-particle, thus allowing inserts larger than 70-90 kb, as obtained by the packaging route. Using this approach, we have recently constructed a 140,000 clone human P1-derived Artificial Chromosome (""""""""PAC"""""""") library with-average inserts of 130 kb. A similar mouse library is now being prepared. The main objective of this proposal is to improve the cloning technology, by creating more versatile vectors, by optimizing the electroshock transformation method and by the development of alternative transformation protocols employing E.coli spheroplasts. The goal is technique development to create second generation libraries with further insert size improvements and containing mammalian expression and replication options. In a later stage, we will utilize the improved procedures for the construction of 5-10 fold redundant libraries for the human genome and for the mouse genome. These libraries will be characterized by stringent criteria to determine clonal stability, levels of cloning artifacts and genomic representation. Once the initial tests are satisfactory, we will make the libraries widely available for genome mapping, DNA sequencing, disease gene cloning and functional studies. This is intended to occur within 4-6 months after the completion of each library. A second objective is to explore technologies for obtaining PACs from existing YAC contigs, either by subcloning approaches or by screening genomic PAC libraries with YAC-derived probes. The focus of the second goal is to make PACs accessible for DNA sequencing efforts, for structure/function studies in somatic cells or transgenic animals and to permit functional gene cloning.
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