Bacterial artificial chromosome libraries for mouse sequencing and functional analysis

Bacterial artificial chromosome (BAC) and Pi-derived artificial chromosome (PAC) libraries providing, a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for ge nomic digestion. A large-insert PAC library was prepared from the 129S6/SvE vTac strain in a bacterial/mammalian shuttle vector to facilitate functiona l gene studies. For genome mapping and sequencing, we prepared BAC librarie s 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 numb ers of clones and the observed average insert sizes, we estimate each libra ry 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 genomi c representation in the three libraries, we analyzed three contigs, each es tablished after screening with a single unique marker. New markers were est ablished fl om the end sequences and screened against all the contig member s 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 ex tensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones w ere conclusively nonchimeric because both end markers or sequences were map ped to the other confirmed contig members. We could not exclude chimerism f or the remaining 41 clones because one or both of the insert termini did no t contain unique sequence to design markers. The low rate of chimerism, sim ilar to 1%, and the low level of detected rearrangements support the antici pated usefulness of the BAC libraries for genome research.