Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B...

Gene therapy studies require techniques that allow alteration of human genomic DNA sequences. Bacterial artificial chromosome cloning systems (BACs/PACs) bridge the gap between vectors with small inserts and yeast artificial chromosomes (YACs). We report the use of a second generation BAC vector, pEBAC, containing eukaryotic selectable markers and combining some of the best features of the BAC, PAC and HAEC systems, into which a 185 kb sequence containing the human beta-globin gene cluster was retrofitted. To permit the introduction of mutations corresponding to those causing human pathology, we have adapted an inducible homologous recombination system for use in E. coli DH10B cells, the host strain for BACs and PACs. Using this system, we have introduced PCR fragments carrying a selectable marker and a reporter gene downstream of the IVS-110 splicing mutation into a specific site within the beta-globin gene sequence. The use of this inducible system minimises the risk of unwanted rearrangements by recombination between repetitive elements and allows the introduction of relevant modifications or reporters at any specific sequence within BACs/PACs in E. coli DH10B cells.