{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/34369"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/34369","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Assessment of Murine Embryo Development Following Electroporation and Microinjection of a Green Fluorescent Protein DNA Construct","abstract":"Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos.","abstract_html":"Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P &lt; 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P &lt; 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P &gt; 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos.","abstract_has_math":false,"creators":["Schmotzer, Carolyn Anne"],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Dairy Science","degree_department":"Dairy Science","school":null,"contributors":[],"advisors":[],"committee_chairs":["Gwazdauskas, Francis C."],"committee_members":["Akers, Robert Michael","Pearson, Ronald E."],"year":2001,"date_issued":"2001-07-31","date_published":"2001-07-31","updated_at":"2026-07-22T22:20:07Z","subjects":["Green Fluorescent Protein","Embryo","Microinjection","DNA","Electroporation"],"languages":[],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-08062001-122413"],"render_values":[{"text":"etd-08062001-122413","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/34369","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Gwazdauskas, Francis C."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Akers, Robert Michael","Pearson, Ronald E."]},{"key":"dc:contributor.department","label":"Department","values":["Dairy Science"]},{"key":"dc:creator","label":"Author","values":["Schmotzer, Carolyn Anne"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T20:42:49Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T20:42:49Z","2002-08-06"]},{"key":"dc:date.issued","label":"Date","values":["2001-07-31"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Dairy Science"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Green Fluorescent Protein","Embryo","Microinjection","DNA","Electroporation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-08062001-122413"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/34369"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:title","label":"Title","values":["Assessment of Murine Embryo Development Following Electroporation and Microinjection of a Green Fluorescent Protein DNA Construct"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Gwazdauskas, Francis C."],"dc:contributor.committeemember":["Akers, Robert Michael","Pearson, Ronald E."],"dc:contributor.department":["Dairy Science"],"dc:creator":["Schmotzer, Carolyn Anne"],"dc:date.accessioned":["2014-03-14T20:42:49Z"],"dc:date.available":["2014-03-14T20:42:49Z","2002-08-06"],"dc:date.issued":["2001-07-31"],"dc:description.abstract":["Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos."],"dc:description.degree":["Master of Science"],"dc:identifier.other":["etd-08062001-122413"],"dc:identifier.uri":["http://hdl.handle.net/10919/34369"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Green Fluorescent Protein","Embryo","Microinjection","DNA","Electroporation"],"dc:title":["Assessment of Murine Embryo Development Following Electroporation and Microinjection of a Green Fluorescent Protein DNA Construct"],"dc:type":["Thesis"],"thesis:degree_discipline":["Dairy Science"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:20:07Z"}