PubMed · 12904662
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Allison F Gillaspy. 2003. Sequencing using fluorescent-labeled nucleotides.. https://doi.org/10.1385/1-59259-409-3%3A195
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Bacterial artificial chromosome (BAC) is widely used to manipulate herpesvirus genome and generate recombinant virus. Here, we developed a new KSHV BACmid, namely as iBAC, by replacing the EGFP with TET3G transactivator under EF1α promoter and inserted Tet response elements in the promoter of RTA in the original KSHV BAC16 clone and characterized KSHV lytic replication in SLK-iBAC cells. SLK-iBAC cells developed more efficient lytic replication and generated more progeny virus than iSLK-BAC16 cells upon the same conditions of doxycycline treatment. Since SLK-iBAC cells only occupied hygromycin selection marker, it is convenient to generate cellular gene knockout via lentivirus-mediated CRISPR-Cas9 or stably express viral or cellular gene via lentivirus followed by antibiotic selection, making iBAC system a better tool to identify cellular targets of viral proteins in the context of virus infection or study the role of viral or cellular genes for KSHV lytic replication and pathogenesis. In addition, iBAC is color-free and can be utilized to track subcellular localization of viral proteins or colocalization between different viral proteins by introducing fusing fluorescent proteins into the BAC backbone. Therefore, the new KSHV iBAC is a powerful inducible tool to study KSHV lytic replication and pathogenesis in cell model.
Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies. Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation. We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC). Using Agrobacterium tumefaciens, these BIBACs can be efficiently transformed to virtually all organisms, including plants, fungi, yeasts and human cells. As the strategy is based on in vivo recombineering and does not depend on restriction sites, it is applicable to any vector. To show the feasibility of the method five BACs, containing 0-75 kb of fungal DNA, were converted into BIBACs. These were subsequently transformed to the plant pathogenic fungus Fusarium oxysporum f.sp. lycopersici and to Aspergillus awamori, a filamentous fungus often used for large-scale protein production. Molecular characterisation of the transformants showed that the BIBACs were efficiently transferred to the fungi and stably integrated into their genomes.
Genome-wide physical maps are crucial to many aspects of advanced genome research. We report a genome-wide, bacterial artificial chromosome (BAC) and plant-transformation-competent binary large-insert plasmid clone (hereafter BIBAC)-based physical map of the soybean genome. The map was constructed from 78001 clones from five soybean BAC and BIBAC libraries representing 9.6 haploid genomes and three cultivars, and consisted of 2905 BAC/BIBAC contigs, estimated to span 1408 Mb in physical length. We evaluated the reliability of the map contigs using different contig assembly strategies, independent contig building methods, DNA marker hybridization, and different fingerprinting methods, and the results showed that the contigs were assembled properly. Furthermore, we tested the feasibility of integrating the physical map with the existing soybean composite genetic map using 388 DNA markers. The results further confirmed the nature of the ancient tetraploid origin of soybean and indicated that it is feasible to integrate the physical map with the linkage map even though greater efforts are needed. This map represents the first genome-wide, BAC/BIBAC-based physical map of the soybean genome and would provide a platform for advanced genome research of soybean and other legume species. The inclusion of BIBACs in the map would streamline the utility of the map for positional cloning of genes and QTLs, and functional analysis of soybean genomic sequences.