BACKGROUND
plasmid dna is recognised as foreign in vertebrate cells because of the abundance and the unmethylated status of cpg dinucleotides in the bacterial sequences.
however, bacterially derived non-replicating minicircle dna, which is created by removal of plasmid backbone dna through intramolecular site-specific recombination within a minicircle producer plasmid, can be engineered to contain predominantly human sequences with a low incidence of immunostimulatory cpg-containing motifs  <cit> .
therefore, minicircle dna is an excellent vector for non-viral gene therapy, where a robust immune response can interfere with gene delivery .
large-scale production of minicircle dna requires a bacterial strain containing a tightly controlled gene for site-specific recombinase, which can be expressed for production of the non-replicating minicircle after the bacterial culture harbouring the minicircle producer plasmid reaches the stationary growth phase.
previously we described the generation of a reca+ escherichia coli strain, mm219cre, containing the chromosomally located cre recombinase gene under the tight control of the arac regulon  <cit> .
a single copy of the cre gene ensures an extremely low leakage level of cre recombinase expression under repression conditions.
untimely cre recombinase expression during expansion of bacterial culture can cause maintenance instability of the minicircle producer plasmids and their premature dissociation into the minicircle and the miniplasmid recombination products with the subsequent loss of the non-replicating minicircle  <cit> .
low mutation load is of utmost importance in the production of dna for human gene therapy protocols.
reca-deficient bacterial strains are often advantageous for production of plasmids with eukaryotic dna sequences because undesirable spontaneous deletion mutations tend to occur with a lower frequency in the reca-deficient background  <cit> .
therefore, we undertook to construct a reca-deficient single-copy cre gene expression system for production of the minicircle vector dna.
to insert the cre gene into the bacterial chromosome we designed a version of the technique for gene placement, which relies on homology-mediated "pop-in, pop-out" of a plasmid with a temperature sensitive mutation in the psc <dig> replication origin  <cit> .
we employed a portable plasmid-born recet recombination module  <cit>  to create transient homologous recombination proficiency in the reca- e. coli strain hb <dig>  thus enabling use of "homology arms" to target an insertion of an expression cassette for the arac controlled cre gene into the chromosomal lacz gene.
RESULTS
integration  of plasmid pbad75cre into the genome of e. coli hb101
the homologous recombination deficient e. coli reca <dig> strain hb <dig> was transformed with the compatible plasmids pbad75cre and pgetrec .
the first plasmid, pbad75cre, provides a cre expression cassette containing the arac regulator gene, which is flanked by two regions of homology to the bacterial lacz gene to enable chromosomal insertion of the cassette via homologous recombination  <cit> .
the plasmid also contains the mutant temperature sensitive replicon from the plasmid psc <dig>  which drives plasmid replication at 30°c, but is shut down in over 99% of cells at 44°c.
the second plasmid, pgetrec  <cit> , is a mobile recombination module with the truncated rece gene and the rect gene under control of the arac regulon.
recet recombinase can drive efficient homologous recombination in a reca- environment  <cit> .
expression of the recet genes was induced by l-arabinose.
subsequent plating of  <dig> ×  <dig> hb <dig> cells harbouring both pbad75cre and pgetrec on lb agar containing x-gal, iptg, cm and ap at 44°c enabled us to isolate  <dig> white lacz- clones containing an insertion  of the entire pbad75cre plasmid in the bacterial lacz gene .
to compare the details of our recet-driven cre gene placement procedure to a reca-driven gene placement protocol described by hamilton et al.
<cit> , we introduced the targeting plasmid pbad75cre into reca+ e. coli strain tb <dig> harbouring plasmid puc <dig>  the chromosomal integration target in this case was the laczΔm <dig> gene expressing a truncated version of β-galactosidase protein, which attained full enzymatic activity in the presence of puc <dig> expressing complementary α-peptide.
after plating  <dig> ×  <dig> cells on x-gal indicator plates at 44°c we isolated  <dig> white lacz- "pop-in" clones .
insertions of the oripsc101-plasmids in the bacterial genome often result in poor growth of the bacteria, thus, simplifying subsequent selection of the clones that underwent excision of the plasmid backbone  <cit> .
we duly observed reduction in colony size of the obtained pbad75cre insertion derivatives of the reca+ strain e. coli tb <dig>  however, the colony size of the pbad75cre insertion derivatives of the reca- strain e. coli hb <dig> remained unchanged compared to the original strain hb <dig>  our direct measurements of od <dig> of the growing liquid bacterial cultures confirmed lack of significant differences between the growth rates of the hb101-based strains.
excision  of the pbad75cre plasmid backbone
to avoid the potential problem of spurious recombinants, the  <dig> lacz- insertion clones were pooled.
the pool was passaged  <dig> times in lb medium supplemented with ap, cm, l-arabinose at 30°c to encourage plasmid excision  from the bacterial chromosome.
the desired excision of the deletion derivative pbad75Δ leaves the arac-cre cassette on the chromosome whilst excision of the entire pbad75cre plasmid reconstitutes the intact chromosomal lacz gene .
to confirm the presence of the lacz+ class of recombinants that underwent excision of the entire pbad75cre plasmid, we plated the passaged cells to produce individual colonies on lb agar supplemented with cm, ap, x-gal and iptg at 30°c.
five lacz+ blue clones were found among  <dig> analysed colonies .
the clones with excised plasmids should acquire inability to grow on selective agar with cm at 44°c because of the loss of oritspsc101-plasmids at this temperature.
to find the clones with "popped-out" plasmids, hamilton et al.
produced individual colonies from the passaged culture at 30°c and screened for cm-sensitivity at 44°c  <cit> .
in our preliminary experiments such screening was difficult to perform because of the substantial background of cells with replicating oritspsc101-plasmids at 44°c.
therefore, we plated the passaged cell population at 44°c to produce individual colonies in conditions favouring loss of the oritspsc101-plasmids.
screening of  <dig> individual colonies  did not reveal any outright cm-sensitive clones but  <dig> partially cm-sensitive clones were spotted  and re-streaked to obtain individual colonies, which were used to screen again for cm-sensitivity.
between  <dig> and  <dig> completely cm-sensitive subclones were isolated for each of the  <dig> clones .
the obtained cm-sensitive subclones were checked for their lacz phenotype on the indicator plates containing lb agar supplemented with iptg and x-gal, and for the presence of the cre gene by pcr .
on this occasion we did not find any lacz+ subclones in which the entire plasmid has been excised and the lacz gene resealed.
all tested cm-sensitive sublcones were lacz- suggesting an insertion in the chromosomal lacz gene.
however, according to pcr analysis,  <dig> partially cm-sensitive clones produced only cre-positive subclones,  <dig> clones produced a mixture of cre-positive and cre-negative subclones and  <dig> clone gave only cre-negative subclones.
in total, among  <dig> tested subclones the cre gene was lost in  <dig> .
lacz- cre- segregants were probably due to plasmid excisions via illegitimate recombination resulting in deletions.
a similar plasmid "pop-out" strategy was followed with the pbad <dig> integration derivatives of the control reca+ e. coli strain tb <dig> .
markedly higher frequencies of excisant recovery were obtained.
lacz+ excisants constituted  <dig>  × 10- <dig> of the passaged cell population .
screening at 44°c allowed to find  <dig> partially cm sensitive clones out of  <dig> ,  <dig> of which were proved to be lacz+ cre-negative,  <dig> clones were lacz- cre-positive and  <dig> clone produced a mixture of lacz+ cre-, lacz- cre- and lacz- cre+ subclones.
to avoid bad luck of picking up a single cre-positive subclone containing a pbad-promoter re-arrangement, the cre-positive subclones were pooled.
the pooled cell population was transformed with the minicircle producer plasmid pfixluc containing mutant loxp <dig> and loxp <dig> sites  <cit>  in conditions of the cre gene repression by d-glucose .
in the absence of selection by ap, the recet-bearing plasmid pgetrec was rapidly lost because it was incompatible with pfixluc, which was maintained by selection with cm.
we obtained  <dig> ap-sensitive clones, grew bacterial cultures in the presence of d-glucose to repress the cre gene, washed the cells and transferred them into the minimal medium supplemented with l-arabinose to induce cre recombinase expression.
formation of the minicircle and the miniplasmid dna was observed in all the tested clones, thereby confirming successful chromosomal placement of the cre gene .
complete expulsion of the recet-bearing plasmid pgetrec from several clones of the new strain hb101cre pfixluc was confirmed by pcr with primers ar1f and re2r, which was specific for the junction between the plasmid-born arac and rece genes .
the inserted arac-cre expression cassette and the entire bacterial lacz::arac-cre locus were amplified by long-range pcr, thus, proving successful insertion of the arac-cre cassette into the lacz gene of the strain hb <dig> .
the wild type sequence of the inserted cre gene was also confirmed .
the resultant strain hb101cre pfixluc was cured of the pfixluc plasmid to allow unimpeded introduction of other minicircle producing plasmids.
finally, the reca- phenotype of the new strain hb101cre was confirmed by the uv-sensitivity test  <cit> .
production and isolation of minicircle dna in reca- bacterial strain hb101cre
six half litre cultures of the reca- strain hb101cre, six half litre cultures of the control reca+ strain mm219cre and one half litre culture of the control reca+ strain tb1cre, all of them harbouring the minicircle producer plasmid pfixluc, were grown under catabolite repression conditions.
the cells from each culture were washed, transferred to minimal medium supplemented with l-arabinose, cultured for additional  <dig> hrs and used to extract supercoiled dna.
formation of the minicircle dna was observed in all three bacterial strains .
the minicircle dna was purified individually from each of the  <dig> hb101cre-derived supercoiled dna preparations and each of the  <dig> mm219cre-derived preparations as described previously  <cit> .
the minicircle dna yields were  <dig> ±  <dig> μg per half a litre culture for hb101cre strain and  <dig> ±  <dig> μg per half a litre culture for mm219cre strain.
surprisingly, there was no considerable difference in the extent of multimerisation in hb101cre-extracted and mm219cre-extracted minicircle dna .
the hb101cre-produced and the mm219cre-produced minicircle dna preparations were used for sequencing through the junction formed by intramolecular cre recombination.
the expected sequence of the newly emerged hybrid site loxp66/ <dig>  and the adjacent sequences were confirmed in both minicircle dna preparations .
discussion
the minicircle dna is an attractive "stealth vector" for human gene therapy because it can be engineered to consist virtually entirely of mammalian sequences with low occurrence of cpg motifs that are recognised as a potent signal by the innate immune system.
plasmid dna for human use is normally produced in reca- bacterial strains  <cit>  because the rate of deletion mutations in recombinant plasmids in reca-deficient environment is often lower than in reca-proficient environment  <cit> .
we describe here an upgrade of our cre-mediated system for minicircle dna production  <cit>  to a reca- status.
the described method of chromosomal gene placement is an adaptation of the method of hamilton et al.
<cit>  to the situation where the bacterial strain to be manipulated is reca-deficient.
the major technical difficulty both in the original version of the procedure and its adaptation in our work is negative selection of bacterial clones with resolved plasmid-chromosome co-integrates.
clearly, any selective pressure towards clones with popped-out plasmids simplifies the required screening.
fortuitously for their experiments, hamilton et al observed, "this method works because the presence of the psc <dig> replication origin in the e. coli chromosome dramatically reduced the growth rate of the cell; psc <dig> co-integrates grew very poorly at 30°c on l-agar containing chloramphenicol".
in our experiments we confirmed this crucial growth retardation phenomenon for the control reca+ strain e. coli tb <dig> and psc101-based plasmid pbad75cre.
in this case we were able to obtain plasmid pop-out clones with a comfortable frequency of  <dig>  × 10- <dig>  however, we did not detect any growth retardation for the bacterial clones harbouring co-integrates of the reca- e. coli hb <dig> chromosome and pbad75cre.
still we were able to recover plasmid pop-out clones, although with a lower frequency of  <dig>  × 10- <dig>
our intention to generate a bacterial strain for production of therapeutic minicircle dna limited the scope of available strategies to achieve the desired combination of a chromosomally located cre gene and the reca mutation.
for example, the required cre-expressing reca- strain could have been created by the exchange of the reca+ allele for a reca- allele in the previously constructed cre-expressing strain mm219cre  <cit>  through generalised transduction by the bacteriophages p <dig> or t4gt <dig> from a srl::tn <dig> reca strain  <cit> .
however, the presence of tn <dig> in the minicircle dna producer strain is undesirable since some human therapeutic genes contain potential mutational hotspots for this element .
thus, cdna for the human cftr gene, the therapeutic gene for cystic fibrosis, has one hotspot consensus sequence and cdna for the human dystrophin gene, the therapeutic gene for dmd, has three such potential hotspots.
similarly, we wished to avoid methods of chromosomal gene placement that directly employ insertions by transposons, bacteriophage mu or is-elements  <cit> .
the remaining options for construction of the new reca- strain included hijacking of the site-specific chromosomal integration systems of the bacteriophages λ, φ <dig> and p <dig>  and also homology-mediated gene targeting enabled by introduction of reca  <cit> , recet or redαβ-expressing plasmids.
here we describe the construction of the reca- minicircle dna producer e. coli hb101cre using transient homologous recombination proficiency created by the plasmid-born recet genes and homology-mediated chromosomal "pop-in, pop-out" of a plasmid with a temperature sensitive replication origin, pbad75cre.
it is not clear why the observed frequency of recombination leading to the excision of the entire pbad75cre plasmid from the lacz gene  was  <dig> times lower than the frequency of recombination leading to the excision of the plasmid backbone pbad75Δ, which left the arac-cre expression cassette in the bacterial chromosome .
using homologous recombination driven by the plasmid-expressed reca protein, some authors observed a strong bias towards one or the other type of "pop-out" recombination among individual plasmid insertions but did not notice any common predisposition among all the plasmid insertions  <cit> .
we did not observe any significant disproportion between recombination classes in reca-driven plasmid pop-out in the control strain tb <dig>  screening of  <dig> colonies yielded  <dig> lacz+ cre-  and  <dig> lacz- cre+ clones .
therefore, we hypothesise that predominant recombination between two proximal regions of homology  can be a feature of recet-driven recombination.
if this is indeed so, this property of recet recombination is extremely advantageous for the selection of insertions into the bacterial chromosome or large plasmids after the "pop-out" step of the "pop-in, pop-out" scheme of recombinogenic engineering .
using the new reca- strain hb101cre we obtained a yield of  <dig> ±  <dig> μg of pure minicircle dna per half a litre of bacterial culture and observed formation of minicircle multimers.
the reca <dig> mutation in the hb101cre strain inhibits homologous recombination.
at the same time, the hybrid loxp66/ <dig> site, which is present on the minicircle mfixluc, contains  <dig> nucleotide substitutions in the  <dig> bp sequence of the wild type loxp site and is known to be a poor substrate for cre recombination  <cit> .
therefore, formation of the minicircle multimers in the hb101cre bacteria, containing the wild type cre gene, does not fit well both the homologous recombination mechanism of multimerisation and the cre-mediated recombination mechanism of multimerisation  <cit> .
formation of the loxp66/ <dig> minicircle multimers in a reca- strain can be attributed to induction of the resident chromosomal recet genes  <cit>  or induction of an additional reca-independent pathway, such as discovered recently  <cit> .
recombinant plasmids are known to change host gene expression patterns in e. coli  <cit>  and so, the minicirlcle producer plasmid pfixluc might have been the trigger for induction of the endogenous recombination genes in our bacterial strain hb101cre.
there are no data to suggest that the multimeric minicircle forms are inferior to the minicircle monomer in terms of gene delivery to mammalian cells.
in fact, the multimeric minicircle forms might be of some benefit for higher levels of transient transgene expression  <cit> .
CONCLUSIONS
in summary, we inserted the arac-cre gene expression cassette into the chromosomal lacz gene of e. coli reca- strain hb <dig> by creating transient homologous recombination proficiency using the plasmid-born recet genes and employing homology-mediated chromosomal "pop-in, pop-out" of a cre plasmid pbad75cre with 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 homology regions was  <dig> times higher than that between the distal homology regions.
we isolated pure minicircle dna, which was generated in reca-deficient background, and showed the presence of the multimeric minicircle forms containing mutant loxp66/ <dig> site 5'-taccgttcgt ataatgtatg ctatacgaac ggta-3', known to be a poor substrate for cre-recombinase.
therefore, we question the view that cre-mediated recombination is the dominant mechanism for formation of minicircle multimers.
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.

 plasmid dna is recognised as foreign in vertebrate cells because of the abundance and the unmethylated status of cpg dinucleotides in the bacterial sequences. however, bacterially derived non-replicating minicircle dna, which is created by removal of plasmid backbone dna through intramolecular site-specific recombination within a minicircle producer plasmid, can be engineered to contain predominantly human sequences with a low incidence of immunostimulatory cpg-containing motifs. therefore, minicircle dna is an excellent vector for non-viral gene therapy, where a robust immune response can interfere with gene delivery. large-scale production of minicircle dna requires a bacterial strain containing a tightly controlled gene for site-specific recombinase, which can be expressed for production of the non-replicating minicircle after the bacterial culture harbouring the minicircle producer plasmid reaches the stationary growth phase. previously we described the generation of a reca+ escherichia coli strain, mm219cre, containing the chromosomally located cre recombinase gene under the tight control of the arac regulon. a single copy of the cre gene ensures an extremely low leakage level of cre recombinase expression under repression conditions. untimely cre recombinase expression during expansion of bacterial culture can cause maintenance instability of the minicircle producer plasmids and their premature dissociation into the minicircle and the miniplasmid recombination products with the subsequent loss of the non-replicating minicircle. low mutation load is of utmost importance in the production of dna for human gene therapy protocols. reca-deficient bacterial strains are often advantageous for production of plasmids with eukaryotic dna sequences because undesirable spontaneous deletion mutations tend to occur with a lower frequency in the reca-deficient background. therefore, we undertook to construct a reca-deficient single-copy cre gene expression system for production of the minicircle vector dna. to insert the cre gene into the bacterial chromosome we designed a version of the technique for gene placement, which relies on homology-mediated "pop-in, pop-out" of a plasmid with a temperature sensitive mutation in the psc replication origin. we employed a portable plasmid-born recet recombination module to create transient homologous recombination proficiency in the reca- e. coli strain hb thus enabling use of "homology arms" to target an insertion of an expression cassette for the arac controlled cre gene into the chromosomal lacz gene.  integration of plasmid pbad75cre into the genome of e. coli hb101 the homologous recombination deficient e. coli reca strain hb was transformed with the compatible plasmids pbad75cre and pgetrec. the first plasmid, pbad75cre, provides a cre expression cassette containing the arac regulator gene, which is flanked by two regions of homology to the bacterial lacz gene to enable chromosomal insertion of the cassette via homologous recombination. the plasmid also contains the mutant temperature sensitive replicon from the plasmid psc which drives plasmid replication at 30°c, but is shut down in over 99% of cells at 44°c. the second plasmid, pgetrec, is a mobile recombination module with the truncated rece gene and the rect gene under control of the arac regulon. recet recombinase can drive efficient homologous recombination in a reca- environment. expression of the recet genes was induced by l-arabinose. subsequent plating of × hb cells harbouring both pbad75cre and pgetrec on lb agar containing x-gal, iptg, cm and ap at 44°c enabled us to isolate white lacz- clones containing an insertion of the entire pbad75cre plasmid in the bacterial lacz gene. to compare the details of our recet-driven cre gene placement procedure to a reca-driven gene placement protocol described by hamilton et al., we introduced the targeting plasmid pbad75cre into reca+ e. coli strain tb harbouring plasmid puc the chromosomal integration target in this case was the laczΔm gene expressing a truncated version of β-galactosidase protein, which attained full enzymatic activity in the presence of puc expressing complementary α-peptide. after plating × cells on x-gal indicator plates at 44°c we isolated white lacz- "pop-in" clones. insertions of the oripsc101-plasmids in the bacterial genome often result in poor growth of the bacteria, thus, simplifying subsequent selection of the clones that underwent excision of the plasmid backbone. we duly observed reduction in colony size of the obtained pbad75cre insertion derivatives of the reca+ strain e. coli tb however, the colony size of the pbad75cre insertion derivatives of the reca- strain e. coli hb remained unchanged compared to the original strain hb our direct measurements of od of the growing liquid bacterial cultures confirmed lack of significant differences between the growth rates of the hb101-based strains. excision of the pbad75cre plasmid backbone to avoid the potential problem of spurious recombinants, the lacz- insertion clones were pooled. the pool was passaged times in lb medium supplemented with ap, cm, l-arabinose at 30°c to encourage plasmid excision from the bacterial chromosome. the desired excision of the deletion derivative pbad75Δ leaves the arac-cre cassette on the chromosome whilst excision of the entire pbad75cre plasmid reconstitutes the intact chromosomal lacz gene. to confirm the presence of the lacz+ class of recombinants that underwent excision of the entire pbad75cre plasmid, we plated the passaged cells to produce individual colonies on lb agar supplemented with cm, ap, x-gal and iptg at 30°c. five lacz+ blue clones were found among analysed colonies. the clones with excised plasmids should acquire inability to grow on selective agar with cm at 44°c because of the loss of oritspsc101-plasmids at this temperature. to find the clones with "popped-out" plasmids, hamilton et al. produced individual colonies from the passaged culture at 30°c and screened for cm-sensitivity at 44°c. in our preliminary experiments such screening was difficult to perform because of the substantial background of cells with replicating oritspsc101-plasmids at 44°c. therefore, we plated the passaged cell population at 44°c to produce individual colonies in conditions favouring loss of the oritspsc101-plasmids. screening of individual colonies did not reveal any outright cm-sensitive clones but partially cm-sensitive clones were spotted and re-streaked to obtain individual colonies, which were used to screen again for cm-sensitivity. between and completely cm-sensitive subclones were isolated for each of the clones. the obtained cm-sensitive subclones were checked for their lacz phenotype on the indicator plates containing lb agar supplemented with iptg and x-gal, and for the presence of the cre gene by pcr. on this occasion we did not find any lacz+ subclones in which the entire plasmid has been excised and the lacz gene resealed. all tested cm-sensitive sublcones were lacz- suggesting an insertion in the chromosomal lacz gene. however, according to pcr analysis, partially cm-sensitive clones produced only cre-positive subclones, clones produced a mixture of cre-positive and cre-negative subclones and clone gave only cre-negative subclones. in total, among tested subclones the cre gene was lost in. lacz- cre- segregants were probably due to plasmid excisions via illegitimate recombination resulting in deletions. a similar plasmid "pop-out" strategy was followed with the pbad integration derivatives of the control reca+ e. coli strain tb. markedly higher frequencies of excisant recovery were obtained. lacz+ excisants constituted × 10- of the passaged cell population. screening at 44°c allowed to find partially cm sensitive clones out of, of which were proved to be lacz+ cre-negative, clones were lacz- cre-positive and clone produced a mixture of lacz+ cre-, lacz- cre- and lacz- cre+ subclones. to avoid bad luck of picking up a single cre-positive subclone containing a pbad-promoter re-arrangement, the cre-positive subclones were pooled. the pooled cell population was transformed with the minicircle producer plasmid pfixluc containing mutant loxp and loxp sites in conditions of the cre gene repression by d-glucose. in the absence of selection by ap, the recet-bearing plasmid pgetrec was rapidly lost because it was incompatible with pfixluc, which was maintained by selection with cm. we obtained ap-sensitive clones, grew bacterial cultures in the presence of d-glucose to repress the cre gene, washed the cells and transferred them into the minimal medium supplemented with l-arabinose to induce cre recombinase expression. formation of the minicircle and the miniplasmid dna was observed in all the tested clones, thereby confirming successful chromosomal placement of the cre gene. complete expulsion of the recet-bearing plasmid pgetrec from several clones of the new strain hb101cre pfixluc was confirmed by pcr with primers ar1f and re2r, which was specific for the junction between the plasmid-born arac and rece genes. the inserted arac-cre expression cassette and the entire bacterial lacz::arac-cre locus were amplified by long-range pcr, thus, proving successful insertion of the arac-cre cassette into the lacz gene of the strain hb. the wild type sequence of the inserted cre gene was also confirmed. the resultant strain hb101cre pfixluc was cured of the pfixluc plasmid to allow unimpeded introduction of other minicircle producing plasmids. finally, the reca- phenotype of the new strain hb101cre was confirmed by the uv-sensitivity test. production and isolation of minicircle dna in reca- bacterial strain hb101cre six half litre cultures of the reca- strain hb101cre, six half litre cultures of the control reca+ strain mm219cre and one half litre culture of the control reca+ strain tb1cre, all of them harbouring the minicircle producer plasmid pfixluc, were grown under catabolite repression conditions. the cells from each culture were washed, transferred to minimal medium supplemented with l-arabinose, cultured for additional hrs and used to extract supercoiled dna. formation of the minicircle dna was observed in all three bacterial strains. the minicircle dna was purified individually from each of the hb101cre-derived supercoiled dna preparations and each of the mm219cre-derived preparations as described previously. the minicircle dna yields were ± μg per half a litre culture for hb101cre strain and ± μg per half a litre culture for mm219cre strain. surprisingly, there was no considerable difference in the extent of multimerisation in hb101cre-extracted and mm219cre-extracted minicircle dna. the hb101cre-produced and the mm219cre-produced minicircle dna preparations were used for sequencing through the junction formed by intramolecular cre recombination. the expected sequence of the newly emerged hybrid site loxp66/ and the adjacent sequences were confirmed in both minicircle dna preparations. discussion the minicircle dna is an attractive "stealth vector" for human gene therapy because it can be engineered to consist virtually entirely of mammalian sequences with low occurrence of cpg motifs that are recognised as a potent signal by the innate immune system. plasmid dna for human use is normally produced in reca- bacterial strains because the rate of deletion mutations in recombinant plasmids in reca-deficient environment is often lower than in reca-proficient environment. we describe here an upgrade of our cre-mediated system for minicircle dna production to a reca- status. the described method of chromosomal gene placement is an adaptation of the method of hamilton et al. to the situation where the bacterial strain to be manipulated is reca-deficient. the major technical difficulty both in the original version of the procedure and its adaptation in our work is negative selection of bacterial clones with resolved plasmid-chromosome co-integrates. clearly, any selective pressure towards clones with popped-out plasmids simplifies the required screening. fortuitously for their experiments, hamilton et al observed, "this method works because the presence of the psc replication origin in the e. coli chromosome dramatically reduced the growth rate of the cell; psc co-integrates grew very poorly at 30°c on l-agar containing chloramphenicol". in our experiments we confirmed this crucial growth retardation phenomenon for the control reca+ strain e. coli tb and psc101-based plasmid pbad75cre. in this case we were able to obtain plasmid pop-out clones with a comfortable frequency of × 10- however, we did not detect any growth retardation for the bacterial clones harbouring co-integrates of the reca- e. coli hb chromosome and pbad75cre. still we were able to recover plasmid pop-out clones, although with a lower frequency of × 10- our intention to generate a bacterial strain for production of therapeutic minicircle dna limited the scope of available strategies to achieve the desired combination of a chromosomally located cre gene and the reca mutation. for example, the required cre-expressing reca- strain could have been created by the exchange of the reca+ allele for a reca- allele in the previously constructed cre-expressing strain mm219cre through generalised transduction by the bacteriophages p or t4gt from a srl::tn reca strain. however, the presence of tn in the minicircle dna producer strain is undesirable since some human therapeutic genes contain potential mutational hotspots for this element. thus, cdna for the human cftr gene, the therapeutic gene for cystic fibrosis, has one hotspot consensus sequence and cdna for the human dystrophin gene, the therapeutic gene for dmd, has three such potential hotspots. similarly, we wished to avoid methods of chromosomal gene placement that directly employ insertions by transposons, bacteriophage mu or is-elements. the remaining options for construction of the new reca- strain included hijacking of the site-specific chromosomal integration systems of the bacteriophages λ, φ and p and also homology-mediated gene targeting enabled by introduction of reca, recet or redαβ-expressing plasmids. here we describe the construction of the reca- minicircle dna producer e. coli hb101cre using transient homologous recombination proficiency created by the plasmid-born recet genes and homology-mediated chromosomal "pop-in, pop-out" of a plasmid with a temperature sensitive replication origin, pbad75cre. it is not clear why the observed frequency of recombination leading to the excision of the entire pbad75cre plasmid from the lacz gene was times lower than the frequency of recombination leading to the excision of the plasmid backbone pbad75Δ, which left the arac-cre expression cassette in the bacterial chromosome. using homologous recombination driven by the plasmid-expressed reca protein, some authors observed a strong bias towards one or the other type of "pop-out" recombination among individual plasmid insertions but did not notice any common predisposition among all the plasmid insertions. we did not observe any significant disproportion between recombination classes in reca-driven plasmid pop-out in the control strain tb screening of colonies yielded lacz+ cre- and lacz- cre+ clones. therefore, we hypothesise that predominant recombination between two proximal regions of homology can be a feature of recet-driven recombination. if this is indeed so, this property of recet recombination is extremely advantageous for the selection of insertions into the bacterial chromosome or large plasmids after the "pop-out" step of the "pop-in, pop-out" scheme of recombinogenic engineering. using the new reca- strain hb101cre we obtained a yield of ± μg of pure minicircle dna per half a litre of bacterial culture and observed formation of minicircle multimers. the reca mutation in the hb101cre strain inhibits homologous recombination. at the same time, the hybrid loxp66/ site, which is present on the minicircle mfixluc, contains nucleotide substitutions in the bp sequence of the wild type loxp site and is known to be a poor substrate for cre recombination. therefore, formation of the minicircle multimers in the hb101cre bacteria, containing the wild type cre gene, does not fit well both the homologous recombination mechanism of multimerisation and the cre-mediated recombination mechanism of multimerisation. formation of the loxp66/ minicircle multimers in a reca- strain can be attributed to induction of the resident chromosomal recet genes or induction of an additional reca-independent pathway, such as discovered recently. recombinant plasmids are known to change host gene expression patterns in e. coli and so, the minicirlcle producer plasmid pfixluc might have been the trigger for induction of the endogenous recombination genes in our bacterial strain hb101cre. there are no data to suggest that the multimeric minicircle forms are inferior to the minicircle monomer in terms of gene delivery to mammalian cells. in fact, the multimeric minicircle forms might be of some benefit for higher levels of transient transgene expression.  in summary, we inserted the arac-cre gene expression cassette into the chromosomal lacz gene of e. coli reca- strain hb by creating transient homologous recombination proficiency using the plasmid-born recet genes and employing homology-mediated chromosomal "pop-in, pop-out" of a cre plasmid pbad75cre with 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 homology regions was times higher than that between the distal homology regions. we isolated pure minicircle dna, which was generated in reca-deficient background, and showed the presence of the multimeric minicircle forms containing mutant loxp66/ site 5'-taccgttcgt ataatgtatg ctatacgaac ggta-3', known to be a poor substrate for cre-recombinase. therefore, we question the view that cre-mediated recombination is the dominant mechanism for formation of minicircle multimers. 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. 
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. using the minicircle producing plasmid pfixluc containing mutant loxp and loxp sites, we isolated pure minicircle dna from the obtained reca- producer strain hb101cre. 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 hb the resultant reca- minicircle dna producer strain hb101cre was used to obtain pure minicircle dna, consisting of monomeric and multimeric minicircle forms. the cre gene expression cassette was inserted into the chromosomal lacz gene by creating transient homologous recombination proficiency in the reca- strain hb 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. 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. 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. 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. minicircle dna is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. 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 times higher than between the distal regions. 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 minicircle dna preparation consisted of monomeric and, unexpectedly, also multimeric minicircle dna forms, all containing the hybrid loxp66/ site 5'-taccgttcgt ataatgtatg ctatacgaac ggta-3', which was previously shown to be an inefficient partner in cre-mediated recombination.
 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 hb 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 times higher than between the distal regions. using the minicircle producing plasmid pfixluc containing mutant loxp and loxp 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/ site 5'-taccgttcgt ataatgtatg ctatacgaac ggta-3', which was previously shown to be an inefficient partner in cre-mediated recombination.  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 hb 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. 