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.