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Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome

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Abstract

Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use.

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Acknowledgments

This work was funded by NIH R01HD068901 and R01HL094585 (PI:AH), the Cystic Fibrosis Foundation (PI:AH), a CABS Award from the Burroughs Wellcome Fund, and an NIH/NIGMS New Innovator Award (DP2 OD008717) (PI:STK). We are grateful to Dr. C. Huxley and Dr. G. Kotzamanis for sharing BAC123s, Dr. C. Courvalin and Dr. S. Goussard for the E. coli BM4573 invasin strain, Dr. C. DiDonato for the murine Smn primers, and also Dr. N. Blackledge and N. Gosalia for assistance.

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Authors and Affiliations

  1. Human Molecular Genetics Program, Lurie Children’s Research Center, Chicago, IL, USA

    Austin E. Gillen & Ann Harris

  2. Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Austin E. Gillen & Ann Harris

  3. Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Catherine A. Lucas & Steven T. Kosak

  4. Harris laboratory formerly in Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Pei Ling Haussecker

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  1. Austin E. Gillen
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  2. Catherine A. Lucas
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  5. Ann Harris
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Correspondence to Ann Harris.

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Gillen, A.E., Lucas, C.A., Haussecker, P.L. et al. Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome. Chromosoma 122, 351–361 (2013). https://doi.org/10.1007/s00412-013-0418-9

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  • DOI: https://doi.org/10.1007/s00412-013-0418-9

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