Engineering Commons^™

High Throughput Screening Of Priming Candidates For Impact On Nonviral Gene Delivery, Albert L. Nguyen

Department of Biological Systems Engineering: Dissertations and Theses

Priming, in the context of nonviral gene delivery, is the treatment of cells with a compound prior to gene transfer that enhances transfection efficiency and/or transgene expression. Essentially, it is the application of an adjuvant approach to gene delivery. Effective transfection strategies may require priming to compete with the efficiency of viral transduction in order to achieve clinically relevant efficiency and expression in vivo. To search for priming compounds, a high throughput screen of the NIH Clinical Collection was performed using 25kDa b-PEI, an EGFP/luciferase plasmid, and HEK293T cells. The EGFP reporter was multiplexed with Hoechst 33342 and Resazurin fluorescence …

Identifying Intracellular Pdna Losses From A Model Of Nonviral Gene Delivery, Timothy Michael Martin, Beata Joanna Wysocki, Tadeusz Antoni Wysocki, Angela K. Pannier

Department of Biological Systems Engineering: Papers and Publications

Nonviral gene delivery systems are a type of nanocommunication system that transmit plasmid packets (i.e., pDNA packets) that are programmed at the nanoscale to biological systems at the microscopic cellular level. This engineered nanocommunication system suffers large pDNA losses during transmission of the genetically encoded information, preventing its use in biotechnological and medical applications. The pDNA losses largely remain uncharacterized, and the ramifications of reducing pDNA loss from newly designed gene delivery systems remain difficult to predict. Here, the pDNA losses during primary and secondary transmission chains were identified utilizing a MATLAB model employing queuing theory simulating delivery of pEGFPLuc …

Temporal Endogenous Gene Expression Profiles In Response To Lipid-Mediated Transfection, Timothy M. Martin, Sarah A. Plautz, Angela K. Pannier

Department of Biological Systems Engineering: Papers and Publications

Background — Design of efficient nonviral gene delivery systems is limited as a result of the rudimentary understanding of the specific molecules and processes that facilitate DNA transfer.

Methods — Lipoplexes formed with Lipofectamine 2000 (LF2000) and plasmid-encoding green fluorescent protein (GFP) were delivered to the HEK 293T cell line. After treating cells with lipoplexes, HG-U133 Affymetrix microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP–) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using …

Temporal Endogenous Gene Expression Profiles In Response To Polymer-Mediated Transfection And Profile Comparison To Lipid-Mediated Transfection, Timothy M. Martin, Sarah A. Plautz, Angela K. Pannier

Department of Biological Systems Engineering: Papers and Publications

Background Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection.

Methods Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP–) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes …