10 research outputs found
Establishing of ES cell lines for a predictable autoregulated expression of transgenes in mice
No full textDie meisten derzeit angewandten Verfahren zur Etablierung transgener Mäuse erfordern ein weitläufiges Screening von Tieren, da die zufällige Integration eines Transgens zu einer, vom Integrationsort abhängigen und daher nicht vorhersagbaren Transgen-Expression führt. Die Wiederverwendung von geeigneten Genorten in ES-Zellen zur Etablierung transgener Mäuse scheint daher sinnvoll. Zu diesem Zweck wurden autoregulierte Expressionskassetten, basierend auf dem TetOff-, TetOn-, E.REXOff- und PIPOff-System, konstruiert, die eine Identifizierung regulierbarer Genorte im Mausgenom und deren Wiederverwendung für eine vorhersagbare, regulierte Expression eines beliebigen Transgens ermöglichen sollten. Die Expressionskapazitäten der Systeme wurden in verschiedenen ES-Zelllinien analysiert. Dabei zeigte sich, dass das TetOn-System ein sehr hohes, das TetOff-System jedoch nur ein niedriges Expressionspotential besitzt. In NIH3T3- und in in vitro differenzierten Zellen wurden jedoch hohe Expressionen mit dem TetOff-System erzielt. Auf der Basis Flp-vermittelter Rekombination wurde eine Kassettenaustauschstrategie entwickelt, die eine Integration eines Transgen-exprimierenden Konstruktes in bereits charakterisierte Genorte, die mit heterospezifischen FRT-Sites markiert sind, erlauben sollte. Diese Strategie wurde in NIH3T3-Zellen getestet und führte zu Zelllinien mit homogener, vorhersagbarer Transgen-Expression.Most of the current procedures for generating transgenic mice require a large scale screening of animals, since random integration of the transgene into the mouse genome results in integration site specific and thus unpredictable transgene expression. The reuse of appropriate integration sites in mouse ES cells for creation of transgenic mice seems to overcome these problems. For this purpose, autoregulated expression cassettes, based on the TetOff, TetOn, E.REXOff and PIPOff system, were designed for screening the mouse genome for inducible chromosomal sites and subsequent reuse of these sites by Flp-mediated recombination for a predictable and regulatable expression of any gene. The expression capacities of these systems were analyzed in different ES cell lines resulting in the observation that the TetOn systems has a very high and the TetOff system only a poor expression potential. However, with the TetOff system high expressions were realized in NIH3T3 cells and in in vitro differentiated cells. For the reuse of transgenic cells a cassette exchange strategy was evolved which should allow the efficient integration of a target construct in a precharacterized locus tagged with heterospecific FRT sites. This strategy was testet in NIH3T3 cells leading to cell lines with homogenous, predictable transgene expression
Nephrocystin-1 Forms a Complex with Polycystin-1 via a Polyproline Motif/SH3 Domain Interaction and Regulates the Apoptotic Response in Mammals
No full textMutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation. © 2010 Wodarczyk et al
RADIO FREQUENCY--MICROWAVE DOUBLE RESONANCE AS A TOOL IN THE ANALYSIS OF MICROWAVE SPECTRA
No full textThis work was supported by the National Science Foundation. S. H. Autler and C. H. Townes, Phys. Rev. 100, 703 (1955).""Author Institution: Department of Chemistry, Harvard University,The practical application of radio frequency-microwave double (RFMDR) as a spectroscopic technique in the analysis of microwave spectra has been investigated. A high-power amplitude-modulated radio-frequency field applied to the Stark septum of a conventional waveguide provides a means of detecting microwave transitions, analogous to the situation in microwave-microwave double resonance (MMDR). The sensitivity of the technique compares favorably with that of MMDR and Stark modulation. Nearly prolate asymmetric top molecules with allowed a-type transitions are especially amenable to the technique due to the presence of favorable radio-frequency transitions between asymmetry doublets. Accidental near degeneracies of dipole-connected rotational levels are also useful. A description of a RFMDR spectrometer using conventional as well as slightly modified Stark cell waveguides will be presented. Limitations and special problems posed by the nature of radio-frequency circuits will be discussed, and examples of applications together with suggestions for other possible uses will be given
A novel mouse model reveals that polycystin-1 deficiency in ependyma and choroid plexus results in dysfunctional cilia and hydrocephalus.
No full textPolycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (approximately 520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases
Nephrocystin-1 forms a complex with polycystin-1 via a polyproline motif/SH3 domain interaction and regulates the apoptotic response in mammals.
No full textMutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation
Polycystin-1 Is Required for Stereocilia Structure But Not for Mechanotransduction in Inner Ear Hair Cells
No full textThepolycystic kidney disease-1(Pkd1) gene encodes a large transmembrane protein (polycystin-1, or PC-1) that is reported to function as a fluid flow sensor in the kidney. As a member of the transient receptor potential family, PC-1 has also been hypothesized to play a role in the elusive mechanoelectrical transduction (MET) channel in inner ear hair cells. Here, we analyze two independent mouse models of PC-1, a knock-in (KI) mutant line and a hair cell-specific inducible Cre-mediated knock-out line. Both models exhibit normal MET channel function at neonatal ages despite hearing loss and ultrastructural abnormalities of sterecilia that remain properly polarized at adult ages. These findings demonstrate that PC-1 plays an essential role in stereocilia structure and maintenance but not directly in MET channel function or planar cell polarity. We also demonstrate that PC-1 is colocalized with F-actin in hair cell stereociliain vivo, using a hemagglutinin-tagged PC-1 KI mouse model, and in renal epithelial cell microvilliin vitro. These results not only demonstrate a novel role for PC-1 in the cochlea, but also suggest insight into the development of polycystic kidney disease.</jats:p
Nephrocystin-1 Forms a Complex with Polycystin-1 via a Polyproline Motif/SH3 Domain Interaction and Regulates the Apoptotic Response in Mammals
No full textMeasurement of the diffractive cross-section in deep inelastic scattering
No full textThis paper presents an analysis of the inclusive properties of diffractive deep inelastic scattering events produced in ep interactions at HERA. The events are characterised by a rapidity gap between the outgoing proton system and the remaining hadronic system. Inclusive distributions are presented and compared with Monte Carlo models for diffractive processes. The data are consistent with models where the pomeron structure function has a hard and a soft contribution. The diffractive structure function is measured as a function of xℙ, the momentum fraction lost by the proton, of β, the momentum fraction of the struck quark with respect to xℙ, and of Q2 in the range 6.3·10-
