Type of Document Master's Thesis Author Dunlap-Brown, Marya URN etd-06252010-151436 Title The In Vitro Transgene Expression and In Vivo Transgene Integration of Condensed DNA Injected into the Cytoplasm of Murine Zygotes Degree Master of Science Department Dairy Science Advisory Committee
Advisor Name Title Gwazdauskas, Francis C. Committee Chair Akers, Robert Michael Committee Member Knight, James W. Committee Member Keywords
Date of Defense 2010-06-11 Availability unrestricted Abstract
Pronuclear stage murine embryos received electrical stimulation in 5, 10, or 20 µs 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 µs pulse length at 100 and 400 V, 10 µs pulse length at 400 V, and 20 µs pulse length at 100, 250, 300 and 400 V. Electroporated embryos that underwent reversible electroporation received 100 V for 5 µs, 400 V for 10 µs, and 250 for 20 µs 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 µg/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 µg/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.
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