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Multigene delivery to mammalian cells and its applications in cell biology

Mansouri, Maysam. Multigene delivery to mammalian cells and its applications in cell biology. 2016, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11781

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Abstract

Multigene delivery systems are emerging as key technologies required in diverse research fields in both academia and industry. Examples include genome editing and synthetic biology, cellular reprogramming and stem cell biology, production of complex proteins in structural biology and many others. Many strategies for assembly and delivery of multigene constructs exist but all of them have their limitations. Genetic constructs are typically introduced into mammalian cells by transient transfection. However, the low efficiency of transient transfection and its limited applicability to a variety of important mammalian cells significantly restrict the utility of this approach.  Viral systems infect many cell types but usually don’t have enough capacity for multiple genes. Therefore, highly efficient delivery of multigene plasmid in a variety of mammalian cells is a challenge which needs to be addressed. We developed MultiPrime, a baculovirus-mediated multigene expression system, allowing simultaneous expression of several genes from a single virus for mammalian cells. MultiPrime is a modular, non-cytotoxic, non-integrating system and works with a variety of promoters. MultiPrime viruses efficiently transduced a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We showed that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression like full length human antibodies in insect and mammalian cells using a single reagent. Also, MultiPrime-infected Zebrafish embryos showed expression of all expected genes in vivo. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells.
The thesis is organized in five chapters. The first chapter provides an introduction to available and common strategies for multigene expression in mammalian cells as well as some of the most important applications of multigene delivery in biological research. The chapters two, three, and four present original research reports covering our established system, an optimized protocol necessary for production and utilization of the system and development of some software for analysis of acquired images by our system. In chapter two, Mansouri et al. (2016) present the main project including establishment of MultiPrime and its diverse applications in cell biology. In chapter three, Mansouri et al. (manuscript in preparation) describe an optimized protocol to generate MultiPrime baculovirus particles that harbor complete gene constructs. In chapter four, Rizk et al. (2014 and 2015) present Squassh, Squassh3C and SquasshAnalyst, three user-friendly software modules that enable segmentation and quantification of subcellular structures of fluorescence microscopy images. In the last Chapter, I discuss and summarize our results and refer to the limitations of our system and I propose possible future directions for genetic manipulation of cells.
Advisors:Ballmer-Hofer, Kurt and Spiess, Martin
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spiess)
Item Type:Thesis
Thesis no:11781
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (110 Seiten)
Language:English
Identification Number:
Last Modified:23 Sep 2016 08:54
Deposited On:23 Sep 2016 08:49

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