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51.
Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats 总被引:10,自引:0,他引:10
Honaramooz A Behboodi E Megee SO Overton SA Galantino-Homer H Echelard Y Dobrinski I 《Biology of reproduction》2003,69(4):1260-1264
Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals. 相似文献
52.
Molecular basis of scrapie strain glycoform variation 总被引:10,自引:0,他引:10
Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs. 相似文献
53.
It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels. 相似文献
54.
SVISS - a novel transient gene silencing system for gene function discovery and validation in tobacco plants 总被引:3,自引:0,他引:3
Gosselé V Faché I Meulewaeter F Cornelissen M Metzlaff M 《The Plant journal : for cell and molecular biology》2002,32(5):859-866
We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family. 相似文献
55.
Poliakov A Hubatsch I Shuman CF Stenberg G Danielson UH 《Protein expression and purification》2002,25(3):363-371
Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni(2+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K(M)) for catalysis and the inhibitory potencies (IC(50) and K(i)) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery. 相似文献
56.
57.
Adorján P Distler J Lipscher E Model F Müller J Pelet C Braun A Florl AR Gütig D Grabs G Howe A Kursar M Lesche R Leu E Lewin A Maier S Müller V Otto T Scholz C Schulz WA Seifert HH Schwope I Ziebarth H Berlin K Piepenbrock C Olek A 《Nucleic acids research》2002,30(5):e21
Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers. 相似文献
58.
Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3. 相似文献
59.
Mineral weathering is the primary source of long-term buffering capacity in soils and is important for forest nutrient sustainability. Regional assessments of weathering rates in Canada and the U.S. have employed an empirical clay-based Soil Texture Approximation (STA) owing to limited data availability, although the STA is rarely calibrated before application to a study area. Soil weathering rates estimated with the STA at 75 sites in Canadian forests (6–367 eq ha?1 year?1) were on average seven times lower than estimates obtained using the PROFILE model and when mineralogy was not available, the Analysis to Mineralogy model and parameter estimation (143–2,119 eq ha?1 year?1). Comparison with a catchment mass balance at a subset of sites in Ontario (n = 19) demonstrated the reliability of PROFILE weathering estimates. A revised (generalized) STA model for total base cation weathering was developed at the 75 study sites to incorporate soil silt content (%) and loss-on-ignition (LOI, %) (BC w = (1.73 + 0.03 · silt ? 0.06 · LOI) · 1,000 · depth). The model performance (R adj 2 = 73%) and relative bias (?1%) suggested that the revised STA may have broad application to forest soils in Canada but may not necessarily be suited to all soil texture classes. 相似文献
60.