Joe Catanese, Baohui Zhao, Eirik Frengen, Chenyan Wu, Xiaoping Guan, Chira Chen, Eugenia Pietrzak, Panayotis A. Ioannou, Julie Korenberg, Joel Jessee 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, Cedar Sinai Medical Center, Los Angeles, CA 90048, Life Technologies, Gaithersburg, MD 20898.
Recently, we have developed procedures for the cloning of large DNA fragments using a bacteriophage P1 derived vector, pCYPAC1 (loannou et al. (1994), Nature Genetics 6: 84-89). A slightly modified vector (pCYPAC2) has now been used to create a 15-fold redundant PAC library of the human genome, arrayed in more than 1,000 384-well dishes. DNA was obtained from blood lymphocytes from a male donor. The library was prepared in four distinct sections designated as RPCI-1, RPCI-3, RPCI-4 and RPCI-5, respectively, each having 120 kbp average inserts. The RPCI-1 segment of the library (3X; 120,000 clones, including 25% non-recombinant) has been distributed to over 40 genome centers worldwide and has been used in many physical mapping studies, positional cloning efforts and in various large-scale DNA sequencing enterprises. Screening of the RPCI-1 library by numerous markers results in an average of 3 positive PACs per autosome-derived probe or STS marker. In situ hybridization results with 250 PAC clones indicate that chimerism is low or non-existing. Distribution of RPCI-3 (3X, 78,000 clones, less than 1% non-recombinants, 4% empty wells) is now underway and the further RPCI-4 and -5 segments (< 5% empty wells) will be distributed upon request. To facilitate screening of the PAC library, we have provided the RPCI-1 PAC library to several screening companies and non-commercial resource centers. In addition, we are now distributing high-density colony membranes at cost-recovery price, mainly to groups having a copy of the PAC library. The combined RPCI-1 and -3 segments (6X) can be represented on 11 colony filters of 22x22 cm, using duplicate colonies for each clone. We are currently generating a similar PAC library from the 129 mouse strain.
To facilitate the additional use of large-insert bacterial clones for functional studies, we have prepared new PAC & BAC vectors with a dominant selectable marker gene (the blasticidin gene under control of the beta-actin promoter), an EBV replicon and an "update feature". This feature utilizes the specificity of Transposon Tn7 for the Tn7att sequence (in the new PAC and BAC vectors) to transpose marker genes, other replicons and other sequences into PACs or BACs. Hence, it facilitates retrofitting existing PAC/BAC clones (made with the new vectors) with desirable sequences without affecting the inserts. The new vector(s) are being applied to generate second generation libraries for human (female donor), mouse and rat.
Supported in part by grants from the Offfice of Health and Environmental Research of the U.S. Department of Energy (#DE-FG02-94ER61883) and the National Center for Human Genome Research, National Institutes of Health (#1R01RG01165).
Return to Table of Contents