Many biopharmaceuticals, already approved for sale or currently under development, are post-translationally modified proteins, such as recombinant monoclonal antibodies or recombinant hormones. These are generally expressed in continuous (stable) mammalian cell lines, which are capable of long-term, commercial-scale production of recombinant proteins of the highest complexity. Yet, the development of a stable cell line capable of expressing heterologous proteins is very costly and can take up to 9–15 months. Therefore, transient gene expression (TGE) in animal cells has become the method of choice for many researchers who wish to obtain small to moderate quantities (1-500 mg) of novel complex recombinant proteins for further functional and structural characterisation within weeks of cDNA discovery. TGE is more cost-effective than the time-consuming establishment of stable cell clones, but a key factor in ensuring that these transient systems have practical application is the availability of efficient and robust transfection agents/methods. While chemical transfection methods currently dominate transient systems, the underlying fundamentals such as the formation of DNA complexes or their mode of function are not fully understood and the characteristics of the complexes and their subsequent ability to transfect cells are variable. This often renders the development of a successful transfection protocol for a new cell line random and researchers frequently have to resort to a trial-and-error approach, testing different media and/or conditions during DNA complex formation, as well as having to fine-tune the cell culture regime pre-, during, and post-transfection. This thesis aimed to explore novel transfection agents and develop DNA complex structure/property—transfection efficiency relationships for these reagents. Two different chemical approaches to transient transfection were investigated: i) a recently suggested inorganic nanoparticle based transfection system which utilises the anion exchange capacity of nanoparticles of a particular family of anionic clays, layered double hydroxides (LDHs), and ii) a modified polyethyleneimine (PEI)-based system, which aimed to reduce the inherent cytotoxicity of high molecular weight (MW) PEI, which is a very effective transfection agent, by constructing high MW mimics from low MW building blocks that are linked to each other via biodegradable linkers such as azomethine groups. While the LDH nanoparticles failed to give satisfactory transfection results for plasmid DNA, they were able to functionally deliver smaller nucleic acids such as siRNA. A mechanism different to that currently accepted for the transfection of mammalian cells with plasmid DNA using LDH nanoparticles as carriers is proposed. The modified polymeric transfection agents were shown to result in significantly less cell death, while maintaining the ability to transfect mammalian cells with almost similar efficiency to that obtained with high MW polyethyleneimine. Generic DNA complex structure/property—transfection efficiency relationships were developed by systematically studying the influence of particle size and zeta potential on transfection results.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.S.)--University of Akron, Dept. of Biomedical Engineering, 2006. / "December, 2006." Title from electronic thesis title page (viewed 12/31/2008) Advisor, Yang Yun; Committee members, Stephanie Lopina, Steven Schmidt; Department Chair, Daniel Sheffer; Dean of the College, George K. Haritos; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 167-181).

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real time PCR. Three cationic methacrylate polymer systems with similar chemical structure were used in this study, namely: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, their transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier compared to both PDMA and PEO-b-PDMA systems. Results from quantitative real-time polymerase chain reaction (real-time PCR), cytotoxicity and Zeta potential measurements showed correlations between the physical properties of the polymers and the efficiencies of cellular uptake of the transgene and transfections. In the case of PEO-b-PDEA system, cytotoxicity was due primarily to the excess polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA complexes to interact with cell effectively was identified as the main barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with other physical characterization techniques can provide greater insights into the transfection barrier at different cellular levels. / Singapore-MIT Alliance (SMA)

A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de alguns desses compostos. Utilizando a estratégia que permite a indução de super-expressão após transfecção gênica, isolamos dois loci relacionados com resistência ao análogo tóxico de purina, tubercidina (TUB). Em um desses locus identificamos um ortólogo do gene TOR (TOxic nucleoside Resistance) em L. (L.) major (TOR-Lm), capaz de conferir altos níveis de resistência a TUB. A identificação e localização cromossomal do segundo locus foi obtida, mas os testes funcionais em presença de TUB não foram tão significativos quanto os obtidos após a transfecção do TOR-Lm. Na segunda parte desta dissertação avaliamos a eficácia da associação de TUB com um inibidor específico do transporte de nucleosídeos em mamíferos, nitrobenziltioinosina (NBMPR), visando reverter a toxicidade de TUB apenas no hospedeiro. Demonstramos que TUB tem uma potente ação anti-parasitária em culturas de Leishmania spp., e que o inibidor NBMPR é capaz de proteger células mamíferas de camundongos infectados da ação tóxica de TUB. / Gene identification associated with drug resistance has contributed to a better understanding of the mechanism of action of anti parasitic compounds. Using transfection and over-expression selection strategy we isolated two loci related with the resistance of tubercidin (TUB), a toxic analog purine. In the first locus we identified an ortholog of the TOR gene (TOxic nucleoside Resistance) in L. (L.) major (TOR-Lm), capable to render wild cells resistance to TUB after transfection and over-expression. Chromosomal location and identification of the second locus was done, but functional tests in the presence of TUB were not as significant as those obtained after TOR-Lm transfection. In the second part of this work, we evaluate the effectiveness of the association of TUB with an inhibitor specific to the mammals nucleoside transport, as nitrobenzylthioinosine (NBMPR), aimed at reversing the TUB toxicity only on the host. We first demonstrate that TUB has a potent anti-parasitic action in cultures of Leishmania spp. Then, we discuss the capacity of the NBMPR inhibitor to protect infected macrophages from the toxic effects of TUB.

abstract: Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge in both of the aforementioned applications is the efficient delivery of siRNA molecules, plasmids or transcription factors to primary cells such as neurons. A majority of the current non-viral techniques, including chemical transfection, bulk electroporation and sonoporation fail to deliver with adequate efficiencies and the required spatial and temporal control. In this study, a novel optically transparent biochip is presented that can (a) transfect populations of primary and secondary cells in 2D culture (b) readily scale to realize high-throughput transfections using microscale electroporation and (c) transfect targeted cells in culture with spatial and temporal control. In this study, delivery of genetic payloads of different sizes and molecular characteristics, such as GFP plasmids and siRNA molecules, to precisely targeted locations in primary hippocampal and HeLa cell cultures is demonstrated. In addition to spatio-temporally controlled transfection, the biochip also allowed simultaneous assessment of a) electrical activity of neurons, b) specific proteins using fluorescent immunohistochemistry, and c) sub-cellular structures. Functional silencing of GAPDH in HeLa cells using siRNA demonstrated a 52% reduction in the GAPDH levels. In situ assessment of actin filaments post electroporation indicated a sustained disruption in actin filaments in electroporated cells for up to two hours. Assessment of neural spike activity pre- and post-electroporation indicated a varying response to electroporation. The microarray based nature of the biochip enables multiple independent experiments on the same culture, thereby decreasing culture-to-culture variability, increasing experimental throughput and allowing cell-cell interaction studies. Further development of this technology will provide a cost-effective platform for performing high-throughput genetic screens. / Dissertation/Thesis / Ph.D. Bioengineering 2012

A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de compostos antileishmania. Pouco se sabe sobre o mecanismo de ação do análogo de purina tubercidina (TUB) em Leishmania. Utilizando a estratégia de superexpressão após transfecção gênica, isolamos um locus de Leishmania (Leishmania) major, de 31 kb, capaz de conferir níveis de resistência quatro vezes maior que o parasita selvagem. Várias deleções desse locus foram geradas e a construção de 3 kb (pSNBR/3kbClaI-EcoRI) também conferiu níveis de resistência quando comparado ao parasita elvagem. Através de análises no genoma de L. (L.) major, localizamos esse locus no cromossomo 31 e, no fragmento de 3 kb, um gene que codifica para uma proteína com função desconhecida até o momento (LmjF.31.2010). Esta proteína foi relacionada com resistência a TUB em todas as linhagens transfectadas analisadas (cosTUB2 e pSNBR/3kbClaI-EcoRI), assim denominamos LmjF.31.2010, de proteína relacionada com resistência a TUB (PRRT). A quantificação relativa de transcritos de mRNA na construção pSNBR/3kbClaI-EcoRI apresentou níveis altos de transcritos da PRRT. Foram gerados ainda mutantes de L. (L.) major e L. (L.) amazonensis resistentes a TUB e estes se apresentaram bem adaptados a concentrações altas de TUB, apresentando razão de resistência maior que 200 vezes, quando comparado com os respectivos parasitas selvagens. A PRRT também foi relacionada na resistência a TUB nos mutantes gerados, pois houve amplificação gênica de prrt. Os resultados obtidos neste trabalho fornecem dados para inferir a importância da PRRT no mecanismo relacionado com resistência a TUB. / The identification of genes associated with resistance to antiparasitic compounds has contributed to a better understanding of the mechanism of action of compounds against Leishmania. Little is known about the mechanism of action of purine analog tubercidin (TUB) in Leishmania. Using a strategy of gene overexpression after transfection, we isolated a locus of Leishmania (Leishmania) major, 31 kb, capable of conferring fold resistance four times greater than the wild type parasite. A set of deletions of this locus were generated and a 3 kb construction (pSNBR/3kbClaI-EcoRI) conferred fold resistance twice than the wild type. Analysis of L. (L.) major genome, located this locus on chromosome 31 and on 3 kb fragment we identified a gene encoding a protein with unknown function (LmjF.31.2010). This protein has been related to TUB resistance in all strains analyzed (cosTUB2 and pSNBR/3kbClaI-EcoRI), so we named mjF.31.2010 of protein related with resistance to TUB (PRRT). Relative quantification of mRNA transcripts in the construction pSNBR/3kbClaI-EcoRI showed high levels of PRRT transcripts.Mutants of L. (L.) major and L. (L.) amazonensis resistant to TUB were also generated and these were well adapted to high TUB concentrations, presenting fol resistance greater than 200 times when compared with their respective wild type. The PRRT was also related to TUB resistance mutants generated by PRRT gene amplification. Despite the high fold resistance presented by TUB resistant mutants, the ratio of expression of these mutant PRRT transfected and wild was similar to the wild type.