Enhancing gene delivery to the mammalian nucleus for applications in viral reverse genetics and human artificial chromosome development.

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dc.contributor.advisorDinman, Jonathan Den_US
dc.contributor.advisorGlass, John Ien_US
dc.contributor.authorBrown, Daviden_US
dc.contributor.departmentCell Biology & Molecular Geneticsen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2017-06-22T05:48:38Z
dc.date.available2017-06-22T05:48:38Z
dc.date.issued2017en_US
dc.description.abstractDelivery of transgenic DNA into mammalian cells is critical to realizing the potential of synthetic biology in advancing gene therapy, construction of entire chromosomes and production of new vaccines and therapeutics in cultured mammalian cells. New synthetic biology techniques such as rapid, inexpensive DNA synthesis have opened the door to engineering biology. However, now the delivery of these synthetic DNA constructs to the nucleus of a living cell is the limiting step in the development of these applications. Living cells possess numerous cellular barriers that a synthetic DNA construct needs to cross to be successfully expressed. In this dissertation, I explore two methods to enhance DNA delivery across the nuclear membrane barrier. First, a plasmid delivery system was developed involving a papillomavirus scaffolding protein that when expressed by the transfected cell line, more consistently delivered plasmids bearing a specific DNA binding site to the nucleus of mammalian cells. This technique enabled us to produce infectious influenza virus more effectively when transfecting mammalian cells with DNA copies of influenza virus genes. These improvements accelerate production of vaccine against influenza virus. Second, we improved an existing method of transferring large DNA molecules cloned in Saccharomyces cerevisiae into cultured cells through polyethylene glycol mediated fusion of the yeast and cultured cells. Creating a reporter yeast strain allowed us to track the percentage of fused cells and the percentage that achieved YCp delivery allowing us to easily optimize the process. By synchronizing recipient cells in mitosis when the nuclear envelope is broken down we increased the delivery efficiency of large YCps ten-fold. This was accomplished by fusing yeast spheroplasts harboring large YCps (up to 1.1 Mb) with cultured cell lines. A statistical design of experiments approach was employed to further boost the vector delivery rate 300-fold to achieve a YCp delivery rate of 1/840 cells. This method was adapted to deliver a 152-kb herpes simplex virus genome cloned in yeast into mammalian cells to produce infectious virus. Finally, we discuss future applications for this technology including the development of human artificial chromosomes and applications in viral reverse genetics for vaccine development.en_US
dc.identifierhttps://doi.org/10.13016/M2CK4F
dc.identifier.urihttp://hdl.handle.net/1903/19334
dc.language.isoenen_US
dc.subject.pqcontrolledMolecular biologyen_US
dc.subject.pqcontrolledCellular biologyen_US
dc.subject.pqcontrolledVirologyen_US
dc.subject.pquncontrolledDNA deliveryen_US
dc.subject.pquncontrolledInfluenzaen_US
dc.subject.pquncontrolledspheroplast fusionen_US
dc.subject.pquncontrolledsynthetic biologyen_US
dc.titleEnhancing gene delivery to the mammalian nucleus for applications in viral reverse genetics and human artificial chromosome development.en_US
dc.typeDissertationen_US

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