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Efficient and precise engineering of a 200 kb β-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system

Abstract

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 β-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 I-110 splicing mutation into a specific site within the β-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.

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Acknowledgements

The authors would like to acknowledge the technical assistance of Ms Anthi Georghiou, Ms Katerina Prountzou and Mr George Christopoulos from the Cyprus Institute of Neurology and Genetics for the screening of the PAC library and the initial characterisation of PAC148/β-globin and of Mr Matthew Newman and Ms Mona El-Masri at the Murdoch Institute for oligonucleotide synthesis. The authors are also grateful to Drs M Messerle, XW Yang, RD Kolodner and GR Smith for useful discussions and to Dr K Murphy for plasmid pTP223. PAI would like to acknowledge the collaboration of J-M Vos in the construction of the pEBAC140 vector, the retrofitting of PAC148/β-globin into this vector and the establishment of a stable cell line in DR cells with EBAC/148β. This work was supported by grants to the Murdoch Institute from the NH&MRC and the Friedreich’s Ataxia Associations of Victoria, New South Wales and Queensland and an Academic Training Award to KN from the Universiti Malaysia Sarawak, Malaysia. KN would like to dedicate this work to the late R Narayanan.

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Narayanan, K., Williamson, R., Zhang, Y. et al. Efficient and precise engineering of a 200 kb β-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther 6, 442–447 (1999). https://doi.org/10.1038/sj.gt.3300901

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