Construction and Characterization of a Large-Fragment Chicken Bacterial Artificial Chromosome Library

Abstract

A large-insert bacterial artificial chromosome (BAC) library has been constructed from male chicken genomic DNA using the new pBeloBAC11 vector. The library was prepared in two parts, such that two-thirds of the BAC library (2976 clones) had an average insert size of 490 kb (80 clones analyzed), after optimization of transformation and HMW DNA size-selection conditions. Fragments of increased average size were cloned by coupling a second size strategy using pulsed-field gel electrophoresis with optimized electroporation that favored transformation ofEscherichia coliDH10B cells with very large plasmids. The initial one-third of this library (1440 clones) was constructed using the standard protocols and had an average insert size of 180 kb (40 clones analyzed). The overall library consists, at present, of 4416 clones with a combined insert size average of 390 kb (ranging from 25 to 725 kb). At least 95% of the BAC clones contain inserts. This is partially due to a second color selection performed with respect to white colonies, as well as to the optimized ligation conditions used. Based on the percentage of clones with inserts and the analysis of insert sizes, we estimate this library to represent a 0.8-fold coverage of the chicken genome. Southern blot analysis and fluorescencein situhybridization were performed to confirm the identity of the BAC inserts with chicken genomic DNA. Analysis of large chicken BAC inserts showed that they were stably propagated for at least 120 cell generations. The results indicate that the BAC system is able to carry stably very large genomic fragments of chicken DNA, this system translating into a powerful tool for physical mapping and positional cloning of the chicken genome.