A novel method for charged macromolecule delivery, called nanoinjection, has been developed at Brigham Young University. Nanoinjection combines micro-fabrication technology, mechanism design, and nano-scale electrical phenomenon to transport exogenous DNA across cell membranes on a nano-featured lance. DNA is electrically accumulated on the lance, precision movements of microelectromechanical systems (MEMS) physically insert the lance into cell, and DNA is electrically released from the lance into the cell. Penetration into the cell is achieved through a two-phase, self-reconfiguring metamorphic mechanism. The surface-micromachined, metamorphic nanoinjector mechanism elevates the lance above the fabrication substrate, then translates in-plane at a constant height as the lance penetrates the cell membranes. In-vitro studies indicate no statistical difference in viability between nanoinjected and untreated mouse zygotes. Pronuclear nanoinjection experiments on mouse zygotes, using microinjection as a control, demonstrate integration and expression of a nanoinjected transgene, and higher rates of zygote survival and pup births than the microinjection control. A new compliant mechanism analysis method, the minimization of potential energy method (MinPE method) is presented to model the equilibrium position of compliant mechanisms with more degrees of freedom (DOF) than inputs, such as a fully-compliant nanoinjector. The MinPE method position and force predictions agree with the method of virtual work and non-linear finite element analyses of under-actuated and underconstrained compliant mechanisms. Additionally, a performance-based comparison is made between quadratic shell finite elements elements and 3-D quadratic solid elements for modeling geometrically non-linear spacial deflection of thin-film compliant mechanisms. The comparison's results suggest the more computationally efficient quadratic shell elements can be used to model spatially deforming thin-film compliant mechanisms. Finally, this dissertation presents preliminary results for a proposed method of DNA transfer called cytoplasm-to-pronucleus nanoinjection. By placing a DNA coated lance into the cytoplasm of a mouse zygote and applying a voltage pulse of sufficient magnitude and duration, pores may open in the pronuclear membranes and DNA may be electrophoretically repelled from the lance. If effective, this process could result in transgenes without having to visualize and physically penetrate into the pronucleus. While embryo survival has been demonstrated under a variety of injection conditions, further study is needed to increase the process' consistency, and to determine if cytoplasm-to-pronucleus nanoinjection can generate transgenic animals.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-4766 |
| Date | 28 June 2011 |
| Creators | Aten, Quentin Theodore |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
Page generated in 0.0021 seconds