No description available.

No description available.

A polymerase chain reaction (PCR) technique was used to detect transgene presence after pronuclear microinjection of mouse zygotes cultured to various stages of development. The transgene was detected in 88% of 1-cell, 88% of 2-cell, 44% of 4- cell, 40% of morula, and 29% of blastocysts. By comparison, the integration frequency for transgenic mice made using the same DNA construct was 22%. After 5 days of in vitro culture, the injected construct was detected in 83% of arrested 1-cell, 85% of arrested 2-cell, and 85% of fragmented embryos. Only 28% of zygotes cultured after microinjection of DNA developed to the blastocyst stage compared to 74% of non-injected zygotes. When DNA buffer alone was injected, 63% of zygotes developed to the blastocyst stage. These data suggest that pronuclear microinjection of DNA is highly detrimental to subsequent embryonic development. Also, most injected DNA that is either unintegrated or that will not be integrated into the genome has been degraded by the blastocyst stage such that it can no longer be detected by PCR. The production of transgenic mice by cytoplasmic injection of DNA mixed with poly-L-lysine is also described. The effects of DNA concentration and stoichiometric ratio of positive charges provided by the polycation to negative charges provided by DNA on transgenic frequency and embryonic viability were studied. The highest transgenic frequency (13% of pups born were transgenic) was obtained when a polylysine/DNA complex having a stoichiometric charge ratio of one to one (equal positive charges as negative charges) at a DNA concentration of 50 ug/ml was used. The transgenic frequency by pronuclear injection of the same DNA construct was 22%. The percentage of zygotes, cultured in vitro, reaching the blastocyst stage which were injected cytoplasmicly was not different (p>0.05) than that of control zygotes that were not microinjected (65% versus 74%, respectively). The percentage of zygotes reaching the blastocyst stage after pronuclear microinjection with DNA at a concentration of 1.5 ug/ml was significantly lower (p<0.05) than control embryos (28% versus 74%, respectively). The overall transgenic pup production efficiency (percent of transgenic pups per embryos transferred) by cytoplasmic injection was 2.4% compared to 3.5% by pronuclear microinjection. / Ph. D.

No description available.

published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

Cardiac fibrosis contributes to heart failure by dramatically impairing cardiac function, increasing patient morbidity and mortality. The primary fibrillar collagen expressed in the heart is type I, and increased collagen synthesis is the hallmark of cardiac fibrosis. Our laboratory has shown that the transcription factor scleraxis is sufficient to regulate the gene encoding collagen Iα2. The present thesis identifies and focuses on three key functions of scleraxis in the heart. First, we show that scleraxis is required for production of the cardiac extracellular matrix. Using in vitro and in vivo models, we observed a significant upregulation/reduction of matrix genes in response to induction/loss of scleraxis gene function respectively. In fact, scleraxis overexpression was sufficient to rescue matrix synthesis in scleraxis-null cells. In a murine model of cardiac pressure overload, scleraxis gene deletion blunted the induction of fibrotic collagen gene expression. Second, we provide evidence that scleraxis governs fibroblast-myofibroblast phenotype transition and fibroblast number. Scleraxis gene induction promoted cardiac myofibroblast phenoconversion while knockdown reduced myofibroblast marker gene expression. Scleraxis exerts direct transcriptional control on the a-smooth muscle actin gene-an established marker of myofibroblasts. Scleraxis null mice exhibited a dramatic reduction in cardiac fibroblast numbers- this is attributed to impairment of the epithelial-to-mesenchymal transition program which was marked by a corresponding loss of mesenchymal markers and increased epithelial markers. Loss-of-function experiments using primary cardiac proto-myofibroblasts recapitulated this paradigm, whereas scleraxis gene induction showed a reciprocal effect on mesenchymal markers. Third, data from this study supports the required role of scleraxis in the TGFb/Smad signaling pathway. Scleraxis is strongly upregulated by the potent pro-fibrotic cytokine TGFb, and works synergistically with the canonical Smad signaling pathway to increase Col1a2 expression by cardiac fibroblasts and myofibroblasts. Smad3 induced expression of the fibrillar collagens – an effect that was significantly attenuated following scleraxis knockdown. Smad3 binding to the Col1a2 gene promoter was significantly reduced in scleraxis null hearts. This study involved a comprehensive series of in vitro and in vivo experiments, and is the first to identify scleraxis as a key regulator of multiple fibroblast functions and a potential future target for therapeutic intervention in cardiac fibrosis. / May 2016