Chromosoma

, Volume 122, Issue 5, pp 351–361

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

  • Austin E. Gillen
  • Catherine A. Lucas
  • Pei Ling Haussecker
  • Steven T. Kosak
  • Ann Harris
Research Article

DOI: 10.1007/s00412-013-0418-9

Cite this article as:
Gillen, A.E., Lucas, C.A., Haussecker, P.L. et al. Chromosoma (2013) 122: 351. doi:10.1007/s00412-013-0418-9

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.

Keywords

Invasin-mediated gene transferCFTR gene expressionBAC recombineeringBAC integration

Supplementary material

412_2013_418_MOESM1_ESM.pdf (1.2 mb)
ESM 1(PDF 1183 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Austin E. Gillen
    • 1
    • 2
  • Catherine A. Lucas
    • 3
  • Pei Ling Haussecker
    • 4
  • Steven T. Kosak
    • 3
  • Ann Harris
    • 1
    • 2
  1. 1.Human Molecular Genetics ProgramLurie Children’s Research CenterChicagoUSA
  2. 2.Department of PediatricsNorthwestern University Feinberg School of MedicineChicagoUSA
  3. 3.Department of Cell and Molecular BiologyNorthwestern University Feinberg School of MedicineChicagoUSA
  4. 4.Harris laboratory formerly in Weatherall Institute of Molecular MedicineUniversity of OxfordOxfordUK