No description available.

No description available.

No description available.

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.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 94-107).

CD4+ T cells playa central role in antitumour immunity; not only do they provide help for the development of CTL recognising tumour antigens but they can also enhance antitumour responses via indirect cytotoxic mechanisms at the tumour site. Since CD4+ T cells recognise the antigen in the form of pep tides presented on MHC class II molecules, attention has been focused in the recent years on the identification of these peptides derived from tumour antigens. Therefore the aim of this study was to identify novel immunogenic peptides derived from tumour antigens where presentation was restricted to human MHC class II HLA-DRI and/or HLADR4 molecules. The adopted strategy consisted in predicting peptides from the tumour antigens p53, gplOO and bcr-abl(b3a2) using computer-assisted algorithms, and immunisation of HLA-DRI transgenic mice with these peptides in order to assess their immunogenicity. Immunogenic peptides in transgenic mice were then tested in human in in vitro T cell sensitisation assays. To determine peptide immunogenicity in mice, a method was optimised using the reported I-Ak-restricted peptides HEL46-61 and HEL1l 9-132. This model was then successfully established in HLA-DRI transgenic mice with the model peptide HA307- 319. Proliferative responses and IFN-y production were observed when the splenocytes of HLA-DRI transgenic mice were re-presented in vitro with the HA307-319 peptide used in immunisation. Dendritic cells (DC) were shown to be better antigen presenting cells (APC) than syngeneic splenocytes in proliferation assays; thus DC were subsequently used as APC in the all experiments. Further characterisation of DC, generated from bone marrow precursors by culture with GM-CSF, demonstrated that day 8 non-adherent cells matured with LPS were optimal for antigen presentation in this experimental setting. Immunisation of HLA-DRI transgenic mice with predicted peptides from p53, gplOO and bcr-abI(b3a2) resulted in HLA-DRlrestricted responses for two novel p53 peptides (p5363-77 and p53108-122) and two bcrabl peptides (bcr-abIGFK11 and bcr-abIATG1 8). Responses were also observed to two novel gp 100 peptides (gp 1 00194-208 and gp 100566-580). This study demonstrated that HLA-DR-restricted responses to novel peptides can be obtained in HLA-DRI transgenic mice. Furthermore, proliferative responses to p5363-77 in a HLA-DRI + donor, to gpl00566-580 in another HLA-DRl+ donor. and to p53108-122 in two HLA- -1- Abstract DR4+ donors demonstrated that these peptides were also immunogenic in human assays. Collectively, these results indicated that peptide immunisation of HLA-DRI transgenic mice could facilitate the identification of novel immunogenic HLA-DRrestricted pep tides from tumour antigens, that allow us to understand further the role of CD4+ in antitumour immunity and improve cancer immunotherapeutic strategies.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy