Many different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems. / text

Based on their highly bioactive properties in membrane phospholipids, there is growing recognition that dietary n-3 polyunsaturated fatty acids (PUFA) may be of significant benefit in the prevention and treatment of many lifestyle related pathologies, however direct evidence is lacking. The fat-1 transgenic mouse, a genetic model of n-3 PUFA enrichment, is a useful tool in nutritional research which has provided enhanced insight into the health effects of lifelong n-3 PUFA exposure. However, the influence of timing of n-3 PUFA exposure on health related outcomes remains unclear. This thesis describes the functional characterization of the novel Cre recombinase dependent inducible fat-1 (iFat-1) transgene. In the presence of Cre, the iFat-1 transgene was found to reduce phospholipid n-6/n-3 PUFA ratios both in vitro (100%) and in vivo (upwards of 70%), suggesting that the iFat-1 transgene has potential application to address temporal effects of n-3 PUFA in health and disease. / Canadian Institutes of Health Research - Frederick Banting and Charles Best Canada Graduate Scholarship, Sun Life Financial

Regensburg, Univ., Diss., 2009.

Univ., Diss., 2006--Giessen

Abstract The complete exon-intron organization of the gene coding for the mouse α1(XIII) collagen chain, Col13a1, was characterized from genomic clones and multiple transcription initiation points were determined. Detailed comparison of the human and mouse genes showed that the exon-intron structures are completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly conserved putative promoter region. The chromosomal location of the mouse gene was determined to be at chromosome 10, band B4, between markers D10Mit5 – (2.3 ± 1.6 cM) – Col13a1 – (3.4 ± 1.9 cM) – D10Mit15. The location of the genes for both the catalytically important α-subunit of prolyl 4-hydroxylase (P4HA) and human type XIII collagen (COL13A1) were previously mapped to 10q21.3-23.1. Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of peptide-bound proline and plays a crucial role in the synthesis of these proteins. The order and transcriptional orientation of the COL13A1 and P4HA was determined. These two genes were found to lie at tail to tail orientation on chromosome 10 and the distance between these genes was determined to be about 550 kbp. To study the function of type XIII collagen we used gene targeting in ES cells to generate a mouse line that carries a mutated type XIII collagen gene. Instead of normal protein, mutant mice express type XIII collagen with an altered amino-terminus in which the cytosolic and the transmembrane domains have been replaced with an unrelated sequence. The homozygous mice are fertile and viable but they show alterations in skeletal muscles, mainly wavy sarcolemma and increased variation in muscle fiber diameter. Ultrastructural studies revealed additional abnormalities such as streaming of z-disks, accumulation and enlargement of mitochondria, and disorganized myofilaments. The basement membranes of the muscle cells showed areas of detachment from the plasma membrane and the fibrillar matrix of the cells was less compact than in control animals. Fibroblasts cultured from mutant mice had normal levels of type XIII collagen but exhibited decreased adhesion to substratum which might be explained by a reduced anchoring strength of the altered protein.

Scleroderma (SSc) is an autoimmune disease characterized by dysfunctional immunity, vasculopathy, and fibrosis of the skin and internal organs. There is a poor prognosis for SSc patients and effective therapeutics have not yet been developed. Many mouse models have been developed, but most fail to recapitulate all of the symptoms seen in SSc patients. In this study we characterize a Fli1flox/flox mouse with CAG Cre under the beta-actin promoter. Based on what has been previously described in mice with deletion of Fli1 in specific cell types, we predicted that global postnatal deletion of Fli1 will result in systemic fibrosis, vasculopathy, and inflammation in these mice. The penetrance of a phenotype was highly variable; however, mice that developed a phenotype displayed disorganized vascular networks, fibrosis and proinflammatory cytokines and chemokines in the skin and lungs after 4 and 8 weeks of Fli1 deficiency. Increased macrophage and dendritic cell populations were observed in the lungs after 8 weeks. Fli1flox/flox CAG Cre mice exhibited hair loss after 5 months of Fli1 deletion. Since our experiments focus mainly on the lungs and skin, more experiments are required to characterize these mice to determine if they can be used as a novel animal model of SSc.

No description available.

No description available.

Mouse polyomavirus is a type species of Polyomaviridae family and serves as model for studying viral infection of human pathogenic polyomaviruses. Minor proteins of viral capsid have been found to be necessary for effective initiation of infection. In order to study their role in the early steps of infection we utilized the novel Cre-LoxP system for production of the viral mutant lacking both minor proteins. Virus produced this way was compared with virus produced by standard method and we found that both systems facilitate production of mutant virus with the comparable quality and quantity. The mutant virus contained reduced amount of viral DNA and formed virions with impaired stability. For further studies of intracellular virion trafficking we prepared virions with genomes modified by thymidine analogues 5- bromo-2'-deoxyuridine (BrdU) and 5-Ethynyl-2'-deoxyuridine (EdU) and optimized the methods for analogue detection. The viral genome become accessible for detection 4 hours post infection. For ultramicroscopic analysis of translocation of virus to the nucleus we used freeze substitution. All this methods will be utilized for detailed study of distinct steps in viral infection. Key words: Mouse polyomavirus, minor proteins,...

Les mécanismes de formation des plaquettes sanguines à partir des mégacaryocytes ne sont pas totalement compris, mais l’environnement médullaire semble y avoir une influence cruciale. Dans ce travail nous montrons que i) les intégrines β3, récepteurs de protéines de matrice extracellulaire, semblent impliquées dans la mégacaryopoïèse et la formation des plaquettes, ii) la différenciation des cellules hématopoïétiques dans un environnement 3D de rigidité comparable à la moelle osseuse améliore la maturation des mégacaryocytes différenciés in vitro et iii) la myosine IIA est impliquée dans la distribution des organelles dans les mégacaryocytes. Parallèlement, Nous avons caractérisé la spécificité d’expression du transgène Pf4-cre pour valider son utilisation dans nos approches expérimentales. Ce travail apporte un éclairage nouveau sur le rôle de la myosine IIA et des intégrines dans les mégacaryocytes et souligne l’influence de la rigidité de l’environnement dans la mégacaryopoïèse. / Megakaryocytes differentiation (megakaryopoiesis) and platelet formation mechanisms are not entirely understood, but the bone marrow environment seems to be crucial in these processes. In this thesis, we show i) that integrin β3, the extracellular matrix protein receptors, are involved in megakaryopoiesis and platelet formation, ii) that recreating a 3D environment of stiffness in the range of that of bone marrow improves the maturation of in vitro differentiated megakaryocytes and iii) a new role for myosin IIA in the cytoplasmic distribution of organelles within the megakaryocyte. As a side-project, we characterized the specificity of expression of the Pf4-cre transgene to validate its use in our experimental approaches. This work enlightens new roles for myosin IIA and integrins in megakaryocytes and indicates that stiffness of the environment influences megakaryopoiesis.