Bacterial Artificial Chromosome Libraries for Mouse Sequencing and Functional Analysis

  1. Kazutoyo Osoegawa1,5,
  2. Minako Tateno1,2,
  3. Peng Yeong Woon1,3,
  4. Eirik Frengen1,4,
  5. Aaron G. Mammoser1,
  6. Joseph J. Catanese1,5,
  7. Yoshihide Hayashizaki2, and
  8. Pieter J. de Jong1,5,6
  1. 1Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263 USA; 2Genome Science Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305 Japan; 3The Wellcome Trust Centre For Human Genetics, University of Oxford, Headington, Oxford OX3 7BN, UK; 4The Biotechnology Centre of Oslo, University of Oslo, N-0349 Oslo, Norway

Abstract

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9–14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, ∼1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.

[The sequence data described in this paper have been submitted to the GenBank data library under accession numbersAQ797173AQ797398.]

Footnotes

  • 5 Present address: Children's Hospital, Oakland Research Institute, 747 Fifty Second Street, Oakland, California 94609-1809 USA.

  • 6 Corresponding author.

  • E-MAIL Pieter.deJong{at}wl.com; FAX (510) 749-4266.

    • Received August 24, 1999.
    • Accepted November 16, 1999.
| Table of Contents

Preprint Server