title: RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA.
abstract: BACKGROUND
  Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions.
  
  
  RESULTS
  We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination.
  
  
  CONCLUSIONS
  Using transient RecET-driven recombination we inserted a single copy of the araC controlled Cre gene into the lacZ gene on the chromosome of E. coli recA- strain HB101. The resultant recA- minicircle DNA producer strain HB101Cre was used to obtain pure minicircle DNA, consisting of monomeric and multimeric minicircle forms. The obtained recA- minicircle DNA producer strain is expected to decrease the risk of undesired deletions within minicircle producer plasmids and, therefore, to improve production of the therapeutic minicircle vectors.
authors.affiliation: Division of Biomedical Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK. o.tolmachov@imperial.ac.uk
authors.forename: Oleg
authors.initials: O
authors.lastname: Tolmachov
authors.forename: Iwona
authors.initials: I
authors.lastname: Palaszewski
authors.forename: Brian
authors.initials: B
authors.lastname: Bigger
authors.forename: Charles
authors.initials: C
authors.lastname: Coutelle
mesh_terms: D002876:Chromosomes, Bacterial; D004270:DNA, Circular / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D016680:Genome, Bacterial; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004270:DNA, Circular; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C084497:RecT protein, E coli; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; C038225:recE protein, E coli