The porcine mammary gland can be used for the production of recombinant proteins by directing a transgene to the mammary gland with a milk protein gene promoter. In order to determine whether or not the protein will be expressed, the animals must be maintained at least through their first lactation. An experiment was performed to determine if hormonal induction of lactogenesis in transgenic virgin pigs could be used as a method for identifying those gilts that are likely to express the recombinant protein during a natural lactation. Mammary development and lactogenesis were induced by administration of subcutaneous implants designed to release 7.1 mg of estradiol-17 beta and 18 mg of progesterone daily for 21 d. Histological analysis of tissue samples before and after the treatment period indicated that mammary secretory tissue underwent dramatic proliferation resulting in a greater degree of alveolar and individual epithelial cell differentiation. The presence of beta-lactoglobulin mRNA was detected in high levels in post-implant tissue samples, and minimally detected in samples cultured in media supplemented with insulin, hydrocortisone, and prolactin. However, protein expression was only detected in the post-implant samples, indicating that beta-lactoglobulin was not maintained well by in vitro culture. The transgene mRNA, recombinant human fibrinogen (A-alpha chain), was detected in all analyzed samples at varying levels. However, the corresponding protein was not detected in any sample, under either reduced or nonreduced conditions. These results indicate that lactogenesis was successfully induced using the hormonal implants. Also, the transgene was activated by the hormonal induction in vivo and in vitro, but the corresponding protein could not be detected. This study indicates that induction of lactogenesis can be used to detect the presence of transgene mRNA in mammary tissue of gilts. However, we cannot conclusively demonstrate that this procedure can be used to identify those gilts that are likely to express the recombinant protein during a natural lactation. / Master of Science

Relaxin is a small peptide hormone that has demonstrated potential therapeutic actions for cardiovascular disease and fibrosis. Additionally, relaxin has demonstrated the ability to protect the heart from injuries caused by ischemia and reperfusion, promote the healing of ischemic ulcers, and counteract allergic responses. The objective of this research was to express fully processed porcine relaxin in transgenic tobacco plants, as an alternative to current methods of producing relaxin. Two recombinant relaxin genes were constructed that contained the patatin signal peptide cDNA fused in frame to prorelaxin cDNA, which was codon-optimized for expression in Nicotiana tabacum, under the control of either the "super" promoter or the dual enhanced cauliflower mosaic virus 35S promoter. Eighteen transgenic tobacco plants were generated that were transformed with the above recombinant genes. Preprorelaxin, mRNA was detected in 12 of the transgenic plants. Fully processed relaxin protein was not found in any tobacco plants that had demonstrated gene expression by northern blot analysis. Preprorelaxin was only identified in extracts from transgenic plants that contained the insoluble protein fraction, as determined by western blot analysis. Additionally, an increased yield of preprorelaxin was identified after incubation of tobacco leaves in an ubiquitin inhibitor. / Master of Science

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Potato yellow dwarf virus (PYDV) is the type member of the genus Nucleorhabdovirus. The virus replicates in the nuclei of infected cells and mature virions accumulate in the perinuclear space after viral cores bud through the inner nuclear membrane. The virus was first described as an extremely destructive pathogen of potato (Solanum tuberosum) and other members of family Solanaceae. There are two different strains of PYDV based on their insect-vector specificity, namely SYDV (sanguinolenta strain) and CYDV (constricta strain). PYDV is considered a model system to study virus-vector relationship, particularly for agriculturally harmful rhabdoviruses. However, very little is known about the molecular aspects and cell biology of PYDV. Preliminary studies showed that infection of transgenic Nicotiana benthamiana plants that constitutively express GFP targeted to endomembranes with SYDV and SYNV (Sonchus yellow net virus, another member of genus Nucleorhabdovirus) results in increased accumulation of GFP and membrane within the infected nuclei, though the pattern of GFP accumulation is completely different for the two viruses. GFP accumulation was found mainly in the external and internal loci of the nucleus in SYDV-infected cells, where as, in the case of SYNV infection, the GFP accumulation was scattered throughout the nucleus of the infected cell. Molecular characterization of SYDV was undertaken to better understand the cellular difference between these two members of Nucleorhabdoviruses. This dissertation describes the determination of the complete nucleotide and ORF (open reading frame) sequences of N (nucleocapsid) and P (Phosphoprotein) gene of SYDV from cDNA clones of both viral genomic and messenger RNAs. Analyses of sequence showed that SYDV-N mRNA contains an 11 nucleotide (nt) untranslated region followed by a 1416 nt ORF encoding a 472 amino acid (aa) protein and P-mRNA contains an 18 nt 5 untranslated region followed by 840 nt ORF encoding a 280 aa protein. Characterization of SYDV-N and P protein using bioinformatic algorithms predict basic hydrophilic and coiled coil regions that may posses the putative nuclear localization signal and protein-protein interaction domain, respectively. Comparison of the SYDV-N ORF with orthologous regions from other plant and animal rhabdoviruses showed statistically significant identity. Phylogenetic analysis based on consensus N-ORFs placed SYDV into the same group with other Nucleorhabdoviruses. Localization studies of SYDV-N and P protein as autofluorescent protein fusions revealed that both proteins are exclusively nuclear localized. Taken together, this dissertation reports a detailed analysis of the biology of SYDV-N and P protein at the molecular and cellular level for the first time towards the long term goal to characterize the entire SYDV genome and to better understand SYDV-host interaction

No description available.

No description available.

No description available.