Download Transgenic bacteria development for minicircle production using

Transgenic bacteria development for minicircle production using ΦC31
recombinase system
Santos, A.A.,1,2; STILHANO, R.S1, HAN, S.W1.
1
Dep. de Biofísica, CINTERGEN, UNIFESP, SP, Brazil; 2 UNISA
A major difficult to the implementation of Gene Therapy (GT) in medical
practice is the construction of suitable vectors that are able to ensure patient
safety with high transfection rate and a sustainable level of gene expression for
therapeutic treatment of disease.
The minicircles vectors (MC) plasmid have reduced size, they are devoid of
bacterial sequences as the origin of replication and antibiotic resistance gene,
allowing a prolonged transgene expression and low immunogenicity.
These vectors are produced through the process of site-specific recombination
mediated by integrases that recognize certain sequences for integration,
inversion or excision depending on the position and orientation of sites of
recombination. One of the difficulties in the production of MC is contamination
with the parental bacterial DNA.
As a way of improving the production of these vectors the objective of
this work was to create a bacterial strain derived from E. coli DH1 expressing
integrase under the control of ΦC31 BAD promoter.
In order to reduce the size of the parental plasmid and increasing the
efficiency of MC production, was developed the DH1ΦC31bacteria using the
homologous recombination method. The plasmid pBADΦC31 was constructed
and the integrase gene was inserted into the site between the LacZ gene, the
vector was transformed into E. coli DH1 (has the LacZ gene in its genome) and
subjected to the protocol of recombination. The product was plated on agar
plates containing LB medium X-Gal and IPTG for growth and differentiation.
The white colonies were analyzed by PCR sequence using specific primers to
integrase ΦC31, the DH1ΦC31 bacteria was amplified by the protocol for the
production of plasmid vectors.
With the construction of DH1ΦC31 will be possible produce MC
efficiently, quickly and free of contamination. Moreover, the bacteria may
decrease the production cost of future drug that is already being successfully
tested in preclinical treatment of various diseases.
Sources of research support: FAPESP