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Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 143 pages. Includes Vita. Includes bibliographical references.

Background: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. Methods: First, we modified the Tet-pLKO-Puro vector to make it easy("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. Results: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. Conclusions: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.

The production of therapeutic recombinant proteins in heterologous systems has gained significance since the last decade. For recombinant proteins that require post-translational modifications (PTMs), mammalian systems are preferred. Chinese hamster ovary (CHO) cells are the mammalian cells of choice for production of recombinant proteins. This is because of their ability to provide correct protein-folding and post-translational modifications, displaying high productivity at large scale, ability to grow in suspension mode at high densities in a serum-free media, incapable of infection by most viruses and their history of regulatory approvals. There is an established state of the art technology for development of CHO cells for recombinant protein production. This technology relies on random integration of the gene of interest and gene amplification process for obtaining high expressing clones. There is a high degree of clonal heterogeneity and instability observed in the screened clones. To overcome the process of random integration, this report describes a lentivirus based screening for search of stable and high expressing integration sites in CHO cells. The integration sites are identified by using nrLAM-PCR (non-restrictive linear amplification mediated PCR) coupled with high throughput sequencing. Lentivirus are chosen as they preferentially integrate within the coding regions rendering the possibility of obtaining stable and high expressing clones. In addition, lentivirus vector is designed to possess landing pad for recombinase mediated cassette exchange of viral sequence with foreign DNA. The report describes a successful cassette exchange reaction but with low efficiency. Genome engineering technologies such as CRISPR/Cas, TALENs can used for targeted gene insertion at integration sites and thus establishing stable and efficient production of recombinant proteins in CHO cells. Additionally, an approach for designing synthetic promoters based on Ef1α promoter architecture has been shown. Synthetic promoters are useful for expression of multi-gene cassettes as they are short in length and provide comparable expression levels to the native mammalian promoter.

RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and short interfering RNAs (siRNAs) have been extensively employed for manipulating gene expression in a wide range of species. The goal for this research was to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. This would demonstrate the utility of RNAi for reducing gene expression in large animal species. To successfully target the myostatin gene for reduction, siRNAs were designed to target the both the bovine and caprine myostatin mRNA sequence. These were then tested for effectiveness in vitro using both an HEK 293T cell line expressing caprine myostatin as well as adult bovine muscle cells. The most effective siRNA, GDF8-1026, was cloned into a lentiviral plasmid and used to transduce bovine fetal fibroblasts for somatic cell nuclear transfer cloning as well as perivitelline injection of in vitro produced bovine embryos. To date, seven pregnancies have been established using these two methods. Concern over the possibility of off-target effects associated with the expression of shRNAs in vivo prompted investigation into tissue-specific expression. Therefore, investigation into the use of a muscle-specific promoter to drive transgene expression was pursued. The bovine myogenin promoter and muscle creatine kinase (MCK) promoter were cloned into a lentiviral plasmid and evaluated in bovine fetal muscle cells and mouse C2C12 cells in vitro for their ability to drive GFP expression. Both promoters demonstrated an increase in GFP intensity at day nine of differentiation when compared to the nontransduced control. The retroviral basis of lentiviral plasmids has raised concern over the possible development of replication competent lentivirus (RCL). Therefore, analysis of tissues from recipients of lentivirus-treated embryos was performed to detect possible RCL. Tissues and blood serum were tested for RCL using p24 ELISA as well as qRT-PCR for the VSV-G gene. To date all tissues tested so far shown no evidence for RCL using these analyses. Analysis of offspring transgenic for an shRNA targeting myostatin will allow confirmation of RNAi as a useful tool for manipulating gene expression in large animal species.

Thèse (Ph. D.)--Université Laval, 2007. / Titre de l'écran-titre (visionné le 5 mai 2008). Bibliogr.

Corneal transplantation is performed to restore vision or to relieve pain in patients with damaged or diseased corneas. However, approximately 40% of corneal allografts fail after 10 years. The most common cause of graft failure is irreversible immunological rejection, primarily mediated by CD4+ T cells, despite the topical application of glucocorticosteroids. The aim of this project was to investigate the anatomic site of antigen presentation during corneal transplantation in the rat, by using a lentiviral vector to express an anti-CD4 antibody fragment at potential sites of antigen presentation, including the donor corneal endothelium, the anterior segment of the eye and the cervical lymph nodes. Dual-gene lentiviral vectors were constructed by inserting the 2A self-processing sequence between two transgenes. This allowed expression of two transgenes within a single open reading frame. In vitro characterisation of the dual-gene vectors was performed in cell culture experiments, which showed that transgenic proteins were expressed at lower levels from dual-gene vectors compared to the expression from single-gene vectors and expression was lowest when the transgene was situated downstream of the 2A self-processing sequence. To locate the anatomic site of antigen presentation during corneal transplantation in rats, a lentiviral vector carrying an anti-CD4 antibody fragment was delivered to the corneal endothelium either immediately prior to corneal transplantation by ex vivo transduction of the donor corneas, or 5 days prior to corneal transplantation by anterior chamber injection into both the recipient and the donor rats. A separate group of recipient rats received intranodal injections of the lentiviral vector carrying an anti-CD4 antibody fragment into the cervical lymph nodes 2 days prior to corneal transplantation. Another group of rats underwent bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Corneal allografts were scored daily for opacity, inflammation and neovascularisation. Expression of the anti-CD4 antibody fragment from transduced tissues was detected using flow cytometry and polymerase chain reaction. Modest, but significant prolongation of corneal allograft survival was experienced by rats that received ex vivo transduction of the donor corneas with a lentiviral vector carrying an anti-CD4 antibody fragment immediately prior to corneal transplantation, but all grafts did eventually reject. Anterior chamber injection of the lentiviral vector carrying the anti-CD4 antibody fragment 5 days prior to corneal transplantation into both recipient and donor eyes did not prolong allograft survival. Intranodal injection of a lentiviral vector carrying an anti-CD4 antibody fragment did not prolong the survival of the corneal allografts, nor did bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Neither expression of the anti-CD4 antibody fragment in the cervical lymph nodes nor the removal of these nodes was able to prolong corneal allograft survival in rats, suggesting that T cell sensitisation could potentially occur elsewhere in the body. However, expression of the anti-CD4 antibody fragment from the donor corneal endothelium was able to prolong corneal allograft survival, suggesting that some antigen presentation might occur within the anterior segment of the eye. Based on the findings described in this thesis and those of others, I propose that antigen presentation in the rat occurs within anterior segment of the eye and within the secondary lymphoid tissues such as the cervical lymph nodes, and that inhibiting antigen presentation at one of these sites will delay graft rejection. However, to completely abolish antigen presentation during corneal transplantation in the rat, I hypothesise that antigen presentation within both the anterior segment of the eye and within the secondary lymphoid tissues must be inhibited.

Thesis (Ph. D.)--Cornell University, March, 2008. / Vita. Includes bibliographical references (leaves 376-419).

Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. [53]-63. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (MSc. (Veterinary Production and Ethology))--University of Pretoria, 2000. / Also available in print format.