Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret

Indexing TNF-α gene expression using a gene-targeted reporter cell line

<p>Abstract</p> <p>Background</p> <p>Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (<it>TNF-α</it>) gene. Drugs known to induce <it>TNF-α </it>expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters.</p> <p>Results</p> <p><it>TNF-α</it>-targeted reporter activity reflected endogenous <it>TNF-α </it>mRNA expression, whereas randomly integrated <it>TNF-α </it>reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), currently used in cancer clinical trials to induce <it>TNF-α </it>gene transcription, was only effective at inducing reporter expression from <it>TNF-α </it>gene-targeted cells.</p> <p>Conclusion</p> <p>We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.</p

Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways

Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family, and is an emerging human pathogenic respiratory virus. In vitro, HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells, we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably, HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase–related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and, more importantly, we identified that two Y-family DNA polymerases, Pol η and Pol κ, are involved in HBoV1 genome amplification. Overall, we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells, which is dependent on the cellular DNA damage and repair pathways

Accurate Mass Determinations in Decay Chains with Missing Energy

Many beyond the Standard Model theories include a stable dark matter candidate that yields missing / invisible energy in collider detectors. If observed at the Large Hadron Collider, we must determine if its mass and other properties (and those of its partners) predict the correct dark matter relic density. We give a new procedure for determining its mass with small error.Comment: 6 figures, 4 page

Adenovirus-Mediated Transfer of the CFTR Gene to Lung of Nonhuman Primates: Biological Efficacy Study

Overview summary Recombinant adenoviruses hold tremendous promise for gene therapies of lung disease in cystic fibrosis (CF). An evaluation of the feasibility and safety of this technology in nonhuman primates is critical in the design of clinical protocols. In a series of two papers, Wilson and colleagues describe an extensive study in baboons designed to evaluate the feasibility and safety of direct instillation of CF transmembrane conductance regulator (CFTR)-expressing adenoviruses into the airway. This paper by Engelhardt et al., addresses the biological efficacy of E1-deleted adenoviruses for gene therapy of CF lung disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63146/1/hum.1993.4.6-759.pd

Lymphoid Enhancing Factor 1 (Lef-1) overexpression in epithelial ovarian, fallopian tube and peritoneal cancer and associations with clinical factors

Lef-1 mRNA levels were statistically elevated in cases of ovarian, fallopian tube or peritoneal cancer when compared to non-cancerous controls. Among cancer cases, levels of Lef-1 were statistically different between stage and histology. Lef-1 overexpression may be predictive of poor overall survival. These findings suggest that Lef-1 overexpression may contribute to ovarian, fallopian tube and peritoneal carcinogenesis, and that further investigation is warranted

On topological charge carried by nexuses and center vortices

In this paper we further explore the question of topological charge in the center vortex-nexus picture of gauge theories. Generally, this charge is locally fractionalized in units of 1/N for gauge group SU(N), but globally quantized in integral units. We show explicitly that in d=4 global topological charge is a linkage number of the closed two-surface of a center vortex with a nexus world line, and relate this linkage to the Hopf fibration, with homotopy $\Pi_3(S^3)\simeq Z$; this homotopy insures integrality of the global topological charge. We show that a standard nexus form used earlier, when linked to a center vortex, gives rise naturally to a homotopy $\Pi_2(S^2)\simeq Z$, a homotopy usually associated with 't Hooft-Polyakov monopoles and similar objects which exist by virtue of the presence of an adjoint scalar field which gives rise to spontaneous symmetry breaking. We show that certain integrals related to monopole or topological charge in gauge theories with adjoint scalars also appear in the center vortex-nexus picture, but with a different physical interpretation. We find a new type of nexus which can carry topological charge by linking to vortices or carry d=3 Chern-Simons number without center vortices present; the Chern-Simons number is connected with twisting and writhing of field lines, as the author had suggested earlier. In general, no topological charge in d=4 arises from these specific static configurations, since the charge is the difference of two (equal) Chern-Simons number, but it can arise through dynamic reconnection processes. We complete earlier vortex-nexus work to show explicitly how to express globally-integral topological charge as composed of essentially independent units of charge 1/N.Comment: Revtex4; 3 .eps figures; 18 page

Ferret and Pig Models of Cystic Fibrosis: Prospects and Promise for Gene Therapy

Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies