Dr. de Jong and colleagues have recently developed new cloning procedures to create large-insert libraries of mammalian genomes in low-copy number bacterial plasmids. The plasmid vector (pCYPAC) is derived from Sternberg's bacteriophage P1 vector (pAdlOSacBII) 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 P1 particle, thus allowing inserts larger than 70-90 kilobases (kb), as obtained by the packaging route. Using this approach, they have recently constructed a 120,000 clone human PAC library with average inserts of 120 kb, which has been widely distributed to over 30 centers worldwide and has been provided at no cost to various commercial clone screening and distribution companies. This ongoing (already funded) effort is directed towards expanding the human library, construction of a similar mouse library and improvements in cloning technology to construct second generation libraries for the human and mouse genomes. The main objective of the current proposal is to apply existing and developing technologies to prepare similar PAC libraries (5-10 hit) for the rat genome. The first and second generation PAC libraries for the rat genome will be characterized by stringent criteria to determine clonal stability, levels of cloning artifacts and genomic representation. The libraries will be made available widely to facilitate positional cloning, DNA sequencing and high-resolution physical mapping. The release will occur once the initial tests are satisfactory and prior to a full characterization. This is anticipated to take place as soon as possible, but will certainly start within 2-4 months after arraying of the library in microtiter dishes. The second objective is to continue the characterization of the libraries with respect to chimerism levels and long-range continuity and provide information on library usage and characteristics to the scientific community. Library information will be provided to the user community through correspondence and through the use of a home page at the World Wide Web.
| Osoegawa, K; Mammoser, A G; Wu, C et al. (2001) A bacterial artificial chromosome library for sequencing the complete human genome. Genome Res 11:483-96 |
| Osoegawa, K; Tateno, M; Woon, P Y et al. (2000) Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. Genome Res 10:116-28 |
| Woon, P Y; Osoegawa, K; Kaisaki, P J et al. (1998) Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library. Genomics 50:306-16 |
| Osoegawa, K; Woon, P Y; Zhao, B et al. (1998) An improved approach for construction of bacterial artificial chromosome libraries. Genomics 52:1-8 |
