The In Vitro Transgene Expression and In Vivo Transgene Integration of Condensed DNA Injected into the Cytoplasm of Murine Zygotes
Pronuclear stage murine embryos received electrical stimulation in 5, 10, or 20 Âµs pulse lengths, and 0, 100, 200, 250, 300 or 400 voltages. Minimal embryo development occurred with 400 V. Irreversible electroporation occurred in embryos electroporated for 5 Âµs pulse length at 100 and 400 V, 10 Âµs pulse length at 400 V, and 20 Âµs pulse length at 100, 250, 300 and 400 V. Electroporated embryos that underwent reversible electroporation received 100 V for 5 Âµs, 400 V for 10 Âµs, and 250 for 20 Âµs and had similar development (P > 0.05) between the best and worst developed groups.
Enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) was condensed with MgCl2 and injected into the cytoplasm of murine zygotes at three concentrations (100, 425 and 625 Âµg/ml). Zygotes injected with the highest concentration had the highest percentages of fluorescing embryos (44%), fluorescing morula and blastocysts (16.7%), and the lowest percentage of mosaicism after 4 d in culture. Five PCR analyses of tail DNA gave conflicting results between 33.3% positive in two or more analyses to 2.8% positive in all five analyses. Southern Analysis detected 2.8% transgenesis. Cytoplasmic injection of linear CMV-EGFP (625 Âµg/ml in water) was 3.7% transgenic. Pronuclear injections produced 7.9% transgenesis.
This research identified a range of reversible electroporation that could easily be verified in vitro with a selectable dye or marker protein and applied in transgenic as well as preclinical treatment models of research. Furthermore this research identifies the benefits and disadvantages of using Mg2+ in DNA condensation and injection buffers.
|dc.rights||I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Virginia Tech or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.||en_US|
|dc.title||The In Vitro Transgene Expression and In Vivo Transgene Integration of Condensed DNA Injected into the Cytoplasm of Murine Zygotes||en_US|
|dc.description.degree||Master of Science||en_US|
|thesis.degree.name||Master of Science||en_US|
|thesis.degree.grantor||Virginia Polytechnic Institute and State University||en_US|
|dc.contributor.committeechair||Gwazdauskas, Francis C.||en_US|
|dc.contributor.committeemember||Knight, James W.||en_US|
|dc.contributor.committeemember||Akers, Robert Michael||en_US|
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