1. ¹Department of Medical Genetics, Childrens Hospital, Ludwig Maximilians University, Munich, Germany
2. ²Unité des Agents Antibactériens, Institut Pasteur, Paris, France
3. ³Centre of Human Genetics, National Institute of Health Dr Ricardo Jorge, Lisboa, Portugal
4. ⁴Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisboa, Lisboa, Portugal
5. ⁵Institute of Human Genetics, Technical University, Munich, Germany
6. ⁶Livestock Biotechnology, Life Sciences Center Weihenstephan, Freising, Germany

Correspondence: C Grillot-Courvalin, Unité des Agents Antibactériens, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, France; D Schindelhauer, Livestock Biotechnology, Life Sciences Center, TUM, Hochfeldweg 1, 85354 Freising-Weihenstephan, Germany

⁷These authors contributed equally to this work

Received 10 February 2005; Accepted 22 May 2005; Published online 23 June 2005.

Abstract

Efficient transfer of chromosome-based vectors into mammalian cells is difficult, mostly due to their large size. Using a genetically engineered invasive Escherichia coli vector, alpha satellite DNA cloned in P1-based artificial chromosome was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic cystic fibrosis transmembrane conductance regulator (CFTR) construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy.