VTechWorks staff will be away for the Thanksgiving holiday beginning at noon on Wednesday, November 27, through Friday, November 29. We will resume normal operations on Monday, December 2. Thank you for your patience.
 

Effect of culture conditions, donor source, and injection site on in vitro development of deoxyribonucleic acid microinjected porcine zygotes

Simple item page
dc.contributor.authorHajdu, Melissa Anneen
dc.contributor.committeechairKnight, James W.en
dc.contributor.committeememberGwazdauskas, Francis C.en
dc.contributor.committeememberVelander, William H.en
dc.contributor.committeememberPearson, Ronald E.en
dc.contributor.departmentAnimal Scienceen
dc.date.accessioned2014-03-14T21:51:52Zen
dc.date.adate2008-12-17en
dc.date.available2014-03-14T21:51:52Zen
dc.date.issued1993-05-05en
dc.date.rdate2008-12-17en
dc.date.sdate2008-12-17en
dc.description.abstractA series of experiments were used to evaluate three culture media and two incubation temperatures for their ability to support development of DNA microinjected porcine zygotes. Development in vitro was compared between embryos collected from postpubertal and prepubertal donors and between embryos injected with DNA into the pronucleus and the cytoplasm. Additionally, embryos were analyzed by the polymerase chain reaction (PCR) for the presence of the transgene. One-cell embryos (n=458) were recovered from 36 postpubertal gilts in Experiment 1. Injected and control embryos were cultured in modified media NCSU-23 (mNCSU-23), NCSU-37 (mNCSU-37), and CZB at 37°C and 38.8°C for 7 d. In Experiment 2, one-cell embryos (n=245) were collected from postpubertal (n=15) and prepubertal (n=14) gilts, microinjected with DNA, and cultured in medium mNCSU-23. Superovulated prepubertal gilts (n=22) were flushed in Experiment 3 to yield 343 one-cell embryos which had DNA injected into the cytoplasm or pronucleus. Whole embryos were assessed by PCR. Mean percentages of embryos developing to the expanded or hatched blastocyst stage in mNCSU-23 and mNCSU-37 did not differ from each other (p>.05), but both were greater than the development in CZB (p<.05). Development was greater at 38.8°C (p<.05) than at 37° C. Microinjection of DNA decreased the developmental percentage (p<.05) from that of non-injected controls. Embryos collected from postpubertal gilts had a higher percentage (68.0 ± 3.4) of expanded and hatched blastocysts than embryos from prepubertal donors (29.0 ± 4.6, p<.05). No difference was seen in development between embryos injected in the pronucleus or cytoplasm (p>. 05), but development for both was less than for control embryos (p<.05). Results of PCR analysis indicated that 40% of the embryos developing to the expanded blastocyst stage were positive for the transgene compared to a rate of 60% positive for degenerate embryos. These studies show that DNA microinjected porcine zygotes can be cultured to the expanded blastocyst stage in media mNCSU-23 and mNCSU-37 at 38.8°C. Microinjection of DNA decreases survival of embryos collected from both postpubertal and prepubertal sources, but postpubertal embryos exhibit a higher rate of development. Cytoplasmic injection does not improve embryo viability in vitro above that of pronuclear injection. Finally, whole embryo analysis by PCR is possible, but cross specificity of human Protein C and whey acidic protein (WAP) oligonucleotides for endogenous porcine DNA is strong and creates difficulty in applying PCR analysis to embryos microinjected with WAP-PC transgenes.en
dc.description.degreeMaster of Scienceen
dc.format.extentvii, 68 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-12172008-063219en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-12172008-063219/en
dc.identifier.urihttp://hdl.handle.net/10919/46281en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V855_1993.H346.pdfen
dc.relation.isformatofOCLC# 28807545en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectmicroinjectionen
dc.subjectporcineen
dc.subject.lccLD5655.V855 1993.H346en
dc.subject.lcshEmbryology -- Cultures and culture mediaen
dc.subject.lcshSwine -- Embryosen
dc.subject.lcshSwine -- Genetic engineeringen
dc.subject.lcshTransgenic animalsen
dc.titleEffect of culture conditions, donor source, and injection site on in vitro development of deoxyribonucleic acid microinjected porcine zygotesen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineAnimal Scienceen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
LD5655.V855_1993.H346.pdf
Size:
2.27 MB
Format:
Adobe Portable Document Format
Download

Collections

Masters Theses