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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)351-361
Number of pages11
JournalChromosoma
Volume122
Issue number5
DOIs
StatePublished - Oct 2013

Keywords

  • BAC integration
  • BAC recombineering
  • CFTR gene expression
  • Invasin-mediated gene transfer

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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