Reversible gene knockdown in mice using a tight, inducible shRNA expression system

RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance was generated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms

Biochemical and functional analyses of chromatin changes at the TCR-&#946; gene locus during CD4<SUP>&#8722;</SUP>CD8<SUP>&#8722;</SUP>to CD4<SUP>+</SUP> CD8<SUP>+</SUP> thymocyte differentiation

Allelic exclusion is the process wherein lymphocytes express Ag receptors from only one of two possible alleles, and is effected through a feedback inhibition of further rearrangement of the second allele. The feedback signal is thought to cause chromatin changes that block accessibility of the second allele to the recombinase. To identify the putative chromatin changes associated with allelic exclusion, we assayed for DNase I hypersensitivity, DNA methylation, and transcription in 100 kb of the TCR-&#946; locus. Contrary to current models, we identified chromatin changes indicative of an active and accessible locus associated with the occurrence of allelic exclusion. Of 11 DNase I hypersensitive sites identified, 3 were induced during CD4&#8722;CD8&#8722;to CD4+ CD8+ thymocyte differentiation, and demethylation and increased germline transcription of the locus were evident. We further examined the role of the most prominently induced site near the TCR-&#946; enhancer (E&#946;) in allelic exclusion by targeted mutagenesis. Two other sites were also examined in New Zealand White (NZW) mice that have a natural deletion in the TCR-&#946; locus. TCR-&#946; gene recombination and allelic exclusion were normal in both mutant mice, negating dominant roles for the three hypersensitive sites in the control of allelic exclusion. The data suggest that alternative cis-regulatory elements, perhaps contained in the E&#946; enhancer and/or in the upstream V&#946; region, are involved in the control of TCR-&#946; allelic exclusion

Enhanced efficiency through nuclear localization signal fusion on phage φC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells

The integrase of the phage ΦC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of ΦC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved ΦC31-integrase as a new tool for genome engineering

&#945;- and &#946;-(BEDT-TTF)2I3 - two modifications with contrasting groundstate properties: insulator and volume superconductor

The &#945;-modification of (BEDT-TTF)2I3 undergoes a metal-to-insulator transition at T = 135 K. Application of hydrostatic pressure shifts the transition temperature to lower values. Above 12 kbar semimetallic behavior is observed, but no superconductivity is detected up to a pressure of 17 kbar and temperatures down to l00 mK. The &#946;-modifcation, on the other hand, exhibits ambient pressure volume superconductivity below Tc = 1.05 K. Measurements of the anisotropy of the diamagnetic shielding- as well as the Meissner-magnetization are presented

Generation of Cre recombinase-specific monoclonal antibodies, able to characterize the pattern of Cre expression in cre-transgenic mouse strains

Transgene-encoded Cre recombinase can target alteration of loxP-tagged genes to specific cell types and developmental stages in mice, depending on the pattern of transgene expression. To facilitate determination of the latter, we have generated monoclonal anti-Cre antibodies which are specific for distinct epitopes on the recombinase and detect Cre both on immunoblots and intracellularly by immunofluorescence. We demonstrate the usefulness of these antibodies by an analysis of Cre expression in mice carrying a cre-transgene under B cell-specific control

New, organic, volume superconductor at ambient pressure

We have observed volume superconductivity in &#946;-(BEDT-TTF)2I3 (&#946;-bis-ethylenedithiolo-tetrathiafulvalene-triiodide) at ambient pressure with a diamagnetic transition temperature of 1.03±0.02 K

Hybrid Embryonic Stem Cell-Derived Tetraploid Mice Show Apparently Normal Morphological, Physiological, and Neurological Characteristics

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo