Matching Items (4)
Description
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.
ContributorsPatel, Chetan (Author) / Muthuswamy, Jitendran (Thesis advisor) / Helms Tillery, Stephen (Committee member) / Jain, Tilak (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2012
Description
Stroke is a devastating disease that affects thousands of individuals each year. Stroke, specifically cerebral ischemia, and immune responses are important areas of study and focus. Previous studies on stroke in mouse models had shown the upregulation of a specific micro-RNA: miR-1224. We hypothesized that miR-1224 was responsible for the regulation of the ST2 receptor protein’s expression. We performed cellular transfection on murine splenocytes with four different miRNAs—miR-1224-mimic, miR-1224-inhibitor, miR-451-mimic, and a control. We predicted that transfection with 1224m would decrease ST2 expression, while transfection with 1224i would increase ST2 expression. Two complete trials were run, and analysis of the results included RT-PCR of both miRNA samples and mRNA samples to confirm transfection and controlled transcription. Reverse transcription and qPCR of miRNA was done in order to confirm that transfection was in fact successful. Reverse transcription and qPCR of the mRNA was done in order to confirm that ST2 mRNA was not altered; this allowed us to attribute any changes in ST2 protein levels to miRNA interactions, as the mRNA levels were consistent. Western blotting was done in order to assess relative protein content. We found that transfection with 1224m slightly decreased ST2 expression and transfection with 1224i slightly increased ST2 expression, however, after assessing the p-values through statistical analyses, neither difference was significant. As such, our hypothesis was rejected as it is not evident that miR-1224 plays a significant role on ST2 gene expression. Future studies are needed in order to analyze alternate protein targets to fully assess the role of miR-1224.
ContributorsReddy, Nihaal (Author) / Holechek, Susan (Thesis director) / Ahmad, Saif (Committee member) / Wood, Kristofer (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Genetic manipulation of human cell lines has widespread applications in biomedical research ranging from disease modeling to therapeutic development. Human cells are generally difficult to genetically engineer, but exogenous nucleic acids can be expressed by viral, chemical, or nonchemical means. Chemical transfections are simpler in practice than both viral and nonchemical delivery of genetic material, but often suffer from cytotoxicity and low efficiency. Novel aminoglycoside antibiotic-derived lipopolymers have been synthesized to mediate transgene delivery to human cells. These polymers are comprised of either paromomycin or apramycin crosslinked with glycerol diglycidylether and derivatized with stearoyl chloride in varying molar ratios. In this work, three previously identified target lipopolymers were screened against a library of human embryonic and induced pluripotent stem cell lines. Cells were transfected with a plasmid encoding green fluorescent protein (GFP) and expression was quantified with flow cytometry 48 hours after transfection. Transfection efficiency was evaluated between three distinct lipopolymers and four lipopolymer:DNA mass ratios. GFP expression was compared to that of cells transfected with commercially available chemical gene delivery reagent controls\u2014JetPEI, Lipofectamine, and Fugene\u2014at their recommended reagent:DNA ratios. Improved transgene expression in stem cell lines allows for improved research methods. Human stem cell-derived neurons that have been genetically manipulated to express phenotypic characteristics of aging can be utilized to model neurodegenerative diseases, elucidating information about these diseases that would be inaccessible in unmanipulated tissue.
ContributorsMehta, Frea (Author) / Brafman, David (Thesis director) / Rege, Kaushal (Committee member) / Chemical Engineering Program (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The use of microelectrode arrays (MEA) to electroporate cells is now a reliable way of transfecting RNA interfering substances with high viability and efficiency. However, as the 50-200 micron electrodes are coated with many cells, there are differences in both viability and efficiency between the outside and inside of the electrode. This is due to the field created by the electrode, which has higher intensities toward the outside and lower intensities toward the middle. In order to get the electric field to spread in a more even manner, an "Anodisc" inorganic membrane seeded with cells was placed on the MEA to act as a buffer to the electric fields. One hundred percent transfection efficiency on live cells was found on one sample, though there were problems encountered along the experimental process that introduced error into the results, some of which included the inability for cells to grow to high levels of confluency on the Anodisc as well as the inverted imaging technique used on the opaque disc.
ContributorsDonnelly, Kyle Robert (Author) / Muthuswamy, Jitendran (Thesis director) / Haynes, Karmella (Committee member) / LaBelle, Jeffrey (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05