RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA

BMC Biotechnology
O TolmachovCharles Coutelle

Abstract

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. 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 usin...Continue Reading

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Citations

Feb 12, 2009·Applied Microbiology and Biotechnology·Diana M Bower, Kristala L J Prather
Oct 29, 2008·Evidence-based Complementary and Alternative Medicine : ECAM·Kourt ChatelainKarl Kingsley
Feb 9, 2012·Advances in Urology·Lidia Mínguez-AlarcónAlberto M Torres-Cantero
May 27, 2008·British Journal of Pharmacology·D J Wells
Mar 28, 2007·Journal of Pharmaceutical Sciences·Kazuhiro HirataYoshinobu Takakura

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Datasets Mentioned

BETA
AF234172

Methods Mentioned

BETA
PCR
electrophoresis

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