Construction and evaluation of a porcine bacterial artificial chromosome library

Abstract

A porcine bacterial artificial chromosome (BAC) library consisting of 103,488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49 x 96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7 x 7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences.

Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1)

“…Swine sequencetagged sites (STSs) were developed from swine expressed sequence tags (ESTs) or swine whole-genome shotgun sequences, which were obtained by BLAST searches with the human gene sequences on the chromosome 9 and then confirmed to be the targeted sequences by using BLAST searches inversely against the human genome (Supplemental Table 1). BAC clones (Suzuki et al 2000) were isolated with swine STS and BAC end sequences were used for chromosome walking. Microsatellite sequences were isolated from BAC clones by using a direct sequencing method reported previously (Fujishima-Kanaya et al 2003).…”

Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp

“…Their particular use is in the study of functional genome segments that are otherwise too large to be cloned into other more conventional vectors. BAC-based libraries of genomic DNA from numerous viruses (1, 4, 8, 29-31, 42, 43) and plant (9,15,27,32,47,53), animal (10,34,50,55), and fungal (20,39,54) species have been generated and are now established technologies in large-scale sequencing projects. Classical and molecular genetic techniques are being used in conjunction with BAC recombinants for the introduction of reporter systems into mammalian organisms, the in vivo complementation of mutations, and in vivo and in vitro reverse genetic technologies that introduce point mutations (25), targeted deletions, or new sequence elements into BAC vectors (21).…”

Construction and preliminary characterization of a river buffalo bacterial artificial chromosome library

“…BAC libraries have been constructed for several species of agricultural importance, including cattle (Cai et al, 1995;Zhu et al, 1999;Buitkamp et al, 2000;Warren et al, 2000;Eggen et al, 2001;Fujisaki et al, 2002), goat , horse (Godard et al, 1998), pig (Rogel-Gaillard et al, 1999Anderson et al, 2000;Suzuki et al, 2000;Fahrenkrug et al, 2001;Jeon et al, 2003;Liu et al, 2010), and sheep (Gill et al, 1999;Vaiman et al, 1999). Although large-insert DNA libraries from closely related ruminant species such as cattle, goat, and sheep have been used for in situ hybridization experiments in buffalo (Di Meo et al, 2005Perucatti et al, 2009), a buffalo library is necessary for whole-genome physical mapping and isolation of genes and gene clusters as well as for the identification of regulatory elements.…”