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Institute for Bioengineering and Biosciences
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Towards Effective Non-viral Gene Delivery Vectors

Towards Effective Non-viral Gene Delivery Vectors | iBB | Scoop.it

Despite very good safety records, clinical trials using plasmid DNA have failed due to low transfection efficiency and brief transgene expression. One of the reasons for this failure is poor plasmid design. In a paper published in Biotechnology and Genetic Engineering Reviews, researchers from iBB-BERG led by Gabriel Monteiro review critical aspects of plasmid design including promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences. The impact of detrimental elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats is also examined. Click on title to learn more.


Photo details: CHO cells transfected with plasmid vector coding for GFP.

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Purification of Minicircles by a Nicking Endonuclease-Assisted Method

Purification of Minicircles by a Nicking Endonuclease-Assisted Method | iBB | Scoop.it

Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from miniplasmid (MP) impurities. In a proof-of-concept study published in Journal of Chromatography A, a team led by Miguel Prazeres from iBB-BERG describe a reproducible process to improve the purification of supercoiled (sc) MCs. The study shows that sc MC can be obtained completely free from nucleic acid impurities by modifying the topology of MP impurities with a nicking endonuclease and subsequently exploring the ability of hydrophonic interaction chromatography to fractionate topoisomers.

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Improvement of DNA Minicircle Production

Improvement of DNA Minicircle Production | iBB | Scoop.it
The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, a team led by Gabriel Monteiro and Miguel Prazeres from iBB-BERG and Kristala Prather from MIT, optimized the 5′ untranslated region (5′-UTR) of parA messenger RNA (mRNA) using a predictive thermodynamic model. The optimized parA gene was then inserted in the chromosome of an E. coli strain with improved arabinose uptake. overall the results show that the improvements made in the 5′-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.The research was developed under the framework of the MIT-Portugal program and is a direct result of Michaela Simcikova's Ph D. thesis in Bioengineering. Click on title to read the publication in Applied Microbiology and Biotechnology.
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