Somatic cell nuclear transfer (SCNT), or cloning, is a form of artificial reproductive technology that can be used to improve economic traits of domestic animals. However, extreme inefficiency of producing viable offspring via this method is a major limitation. An aggressive immune response at the maternal-fetal interface is an important reason for SCNT pregnancy loss. The goal of this project was to investigate the molecular mechanisms of immune-mediated miscarriage in cloned cattle pregnancies. Many publications hint that immune-mediated miscarriage is associated with abnormal MHC-I expression in the placenta. The regulation of bovine MHC-I genes was systematically studied to identify the cause of abnormal MHC-I expression during immune-mediated miscarriage. We also produced cloned pregnancies to study immune- mediated pregnancy loss. MHC-I and cytokines involved in proinflammatory responses were highly expressed in the placental trophoblast cells of cloned fetuses and in the uterine endometrium of recipients carrying MHC-I incompatible fetuses, respectively, suggesting that MHC-I compatibility between fetus and surrogate mother is important for the success of animal cloning. The results from this research not only reveal the cause of high pregnancy loss in cloned animals but also provide molecular clues to prevent immune-mediated miscarriage in cattle and potentially in human clinics.

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 90-119).

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 96-108).

There has been an increase in rates of chromosomal abnormalities in newborns as a result of reproductive aging. For the past decades, a lot of effort has been placed on identifying pregnancies at risk of genetic defects. Conventional prenatal genetic diagnosis is achieved by invasive procedures that have been associated with an increased risk of pregnancy loss. This has led the researchers to explore the use of non-/minimally invasive techniques for prenatal diagnosis. Trophoblasts are known to be shed from regressing chorionic villi into the lower uterine pole of pregnant women during the first trimester. These cells are trapped within cervical mucus, which can be retrieved with a cytobrush. By using human leukocyte antigen G (HLA-G) and cytokeratin-7 (CK7) as trophoblast markers, this study aims to investigate the possibility of isolating individual fetal trophoblast from transcervical samples for genetic diagnosis. 195 healthy pregnant women requesting for legal termination of pregnancy (TOP) were recruited in this study. Transcervical cells were collected from them with the use of a cytobrush before TOP. HLA-G+ or CK7+ cells were then isolated by a combination of mucolytic action, fluorescent immunohistochemistry, and micromanipulation. The origin of these cells was subsequently investigated by either fluorescent in situ hybridization (FISH) or allelic profiling by quantitative fluorescent polymerase chain reaction (QF-PCR) based on chromosome 16, chromosome X, amelogenin gene and sex determining region Y (SRY) gene. This study first demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR. Cells expressing HLA-G and CK7 were also identified among transcervical cells. Immunopositive cells were isolated by micromanipulation under fluorescent microscopy. One isolated cell expressing CK7 was shown to inherit paternal allele at a locus on chromosome 16, suggesting the possible fetal origin of this cell. However, this study was still hampered by a number of technical factors. Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable manner. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

No description available.

Since changes in chromosome morphology contribute to the knowledge of evolution as well as to chromosome dynamics, this study looks specifically at one chromosome compared in twelve different species of Perissodactyls: Equus caballus (ECA), E. przewalskii (EPR), Equus africanus somaliensis (EAF), E. asinus (EAS), E. hemionus onager (EHO), E. h. kulan (EHK), E. h. kiang (EKI), E. zebra hartmannae (EZH), E. grevyi (EGR), E. burchelli (EBU), Tapirus indicus (TIN), and Rhinoceros unicornis (RUN). While chromosome morphology studies have been done in some of the extant equids, none have followed the evolution of this chromosome, homologous to Equus caballus chromosome 20 (ECA20), which contains the major histocompatibility complex (MHC). The gene order on the chromosome arm homologous to human chromosome six in most Equidae is reversed with respect to the centromere in comparison to humans. Multicolor fluorescence in situ hybridization was used to show that four probes from ECA20 hybridized to ECA20 (control), SWA5, EAS8, EHO16, EHK14, EKI16, EZH10, EGR11, EBU13, TIN4, and one of RUN12, 14, 15, or 22. The order for the four genes in the horses, zebras, and rhinoceros were as follows: cen-EDN1-MHC-ITPR3-MUT. Hybridization to the ass and tapir chromosomes displayed a possible neocentromere formation. It is apparent the chromosome has gone through several morphological changes while undergoing speciation in the Equidae, yet the overall gene order is conserved.

2. The long range DNA organisation of the class II and class III regions in eight HLA homozygous cell lines has been analysed using PFGE. Comparison of the size of the BssHII restriction fragment observed for these cell lines and five individuals possessing one to three C4 genes, shows that the organisation of the C4 genes on each chromosome can be deduced from a single PFGE experiment. Outside of the C4 and 21-OHase loci the class III region shows a highly invariant structure, with no detectable differences in the amount of DNA present. Moreover the class III region is rich in CpG-islands, one of which has been characterised, and contains at least thirteen new genes. However, in the class II region, two differences between common haplotypes have been found. The DRw52-related haplotypes have the same DNA organisation. DR2 haplotypes possess 20-30 kb more DNA in the DRB region. DRw53 haplotypes have 100-130 kb more DNA than DRw52-related haplotypes in the region containing the DRB and DQA genes.