A novel approach for regulated trnasgene expression in mammalian cells

A novel ‘screen and insert’ strategy to improve the ease, efficiency and reproducibility with which tightly regulated transgenes can be generated is tested in human cell lines. The strategy involves firstly identifying and characterising by flow cytometry those rare clones whose integration site allows optimal regulation of a reporter gene (encoding the Green Fluorescent Protein, GFP) by the tetracycline (Tet- OFF) inducible system. CVe-mediated site-specific recombination between two mutant loxP sites (lox71 and lox66) is then used to insert any gene of interest (for testing purposes the luciferase ORF was used) downstream of the tightly regulated promoter in such clones. To establish the ‘screen and insert’ approach in cell culture the human fibrosarcoma cell line, HT1080, and the telomerase immortalised retinal epithelial cell line, hTERT-RPEl, were used. The low frequency of Oe-mediated insertion (~1/105 of transfected cells) requires a method of selection for isolating clones and two methods are described. The first (System One) involves insertion of a gene of interest (GOI) which is linked to an IRES-hygromycin cassette to enable selection in hygromycin for the insertion event. A limitation of this strategy, however, is that the GOI must be expressed whilst selection takes place and this may be undesirable for certain GOIs (e.g. when a gene’s product is toxic). The second method (System Two) avoids this limitation by taking advantage of flp-mediated site-specific excision to delay GOI expression until selection for recombination is complete. The utility of the validated ‘screen and insert’ approach is demonstrated by use of the RAD52 gene (involved in homologous recombination and DNA repair) and the I-Scel endonuclease as GOIs. The resulting cell lines will be used to characterise the deleterious effects of RAD52 and I-Scel expression on genome stability and cell viability.Open acces

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

<p>Abstract</p> <p>Background</p> <p>The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites.</p> <p>Results</p> <p>To circumvent these problems we have developed a "screen and insert" strategy in which clones carrying a transgene for a fluorescent reporter are first screened for those with optimal induction characteristics. Site-specific recombination (SSR) is then be used repeatedly to insert any new transgene at the reporter transgene locus of such clones so that optimal induction characteristics are conferred upon it. Here we have tested in a human fibrosarcoma cell line (HT1080) two of many possible implementations of this approach. Clones (e.g. Rht14-10) in which a GFP reporter gene is very stringently regulated by the tetracycline (tet) transactivator (tTA) protein were first identified flow-cytometrically. Transgenes encoding luciferase, I-<it>Sce</it>I endonuclease or Rad52 were then inserted by SSR at a <it>LoxP </it>site adjacent to the GFP gene resulting stringent tet-regulated transgene expression. In clone Rht14-10, increases in expression from essentially background levels (+tet) to more than 10⁴-fold above background (-tet) were reproducibly detected after Cre-mediated insertion of either the luciferase or the I-<it>Sce</it>I transgenes.</p> <p>Conclusion</p> <p>Although previous methods have made use of SSR to integrate transgenes at defined sites, none has effectively combined this with a pre-selection step to identify integration sites that support optimal regulatory characteristics. Rht14-10 and similar HT1080-derived clones can now be used in conjunction with a convenient delivery vector (pIN2-neoMCS), in a simple 3-step protocol leading to stringent and reproducible transgene regulation. This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes.</p

Exceptional Retreat of Kangerlussuaq Glacier, East Greenland, Between 2016 and 2018

Kangerlussuaq Glacier is one of Greenland’s largest tidewater outlet glaciers, accounting for approximately 5% of all ice discharge from the Greenland ice sheet. In 2018 the Kangerlussuaq ice front reached its most retreated position since observations began in 1932. We determine the relationship between retreat and: (i) ice velocity; and (ii) surface elevation change, to assess the impact of the retreat on the glacier trunk. Between 2016 and 2018 the glacier retreated ∼5 km and brought the Kangerlussuaq ice front into a major (∼15 km long) overdeepening. Coincident with this retreat, the glacier thinned as a result of near-terminus acceleration in ice flow. The subglacial topography means that 2016–2018 terminus recession is likely to trigger a series of feedbacks between retreat, thinning, and glacier acceleration, leading to a rapid and high-magnitude increase in discharge and sea level rise contribution. Dynamic thinning may continue until the glacier reaches the upward sloping bed ∼10 km inland of its current position. Incorporating these non-linear processes into prognostic models of the ice sheet to 2100 and beyond will be critical for accurate forecasting of the ice sheet’s contribution to sea level rise

The Grizzly, September 15, 2016

Board Chair Marcon Resigns Amid Controversy • Meet the Interim Board Chair • Black Girl Dangerous Comes to Speak at Ursinus • Student Work Hits the Stage • A Creative Approach to Raising Awareness • Opinions: Choose the America You Wish to be a Part of ; Students\u27 Guide to Weekends at Reimert • Field Hockey Off to a Hot Start, Looking for Redemption • You Bend \u27Em, We Mend \u27Em: The Life of an Athletic Trainerhttps://digitalcommons.ursinus.edu/grizzlynews/1648/thumbnail.jp

Hubungan Asupan Protein, Seng, Zat Besi, Dan Riwayat Penyakit Infeksi Dengan Z-score Tb/u Pada Balita

Latar Belakang : Masalah gizi yang paling banyak ditemukan pada anak di Indonesia adalah stunting, Indikator untuk menilai stunting berdasarkan pada Indeks Tinggi Badan menurut Umur (TB/U) dengan ambang batas (Z-score) &lt;-2 Standart Deviasi (SD). Several micronutrients are required for adequate growth among children. However, it has been unclear as to which nutrient deficiencies contribute most often to growth faltering in populations at risk for poor nutrition and poor growth. Inadequate intakes of dietary energy and protein and frequent infections are well-known causes of growth retardation (3–5). However, the role of specific micronutrient deficiencies in the etiology of growth retardation has gained attention more recently (6–8). Tujuan : Mengetahui hubungan antara asupan protein, seng, zat besi, dan penyakit infeksi terhadap indeks z-score TB/U pada Balita usia 24-59 bulan.Metode : Penelitian observasional dengan pendekatan cross sectional di Kelurahan Jangli Semarang, jumlah sampel 61 Balita usia 24-59 bulan, dipilih dengan simple random sampling. Data yang dikumpulkan meliputi: identitas sampel, berat badan, tinggi badan, riwayat asupan makan, dan riwayat penyakit infeksi. Berat badan diukur menggunakan timbangan digital dan tinggi badan diukur menggunakan microtoise. Asupan protein, seng, zat besi, dan riwayat penyakit infeksi diperoleh dari food frequency questionairre semi-kuantitatif. Data dianalisis dengan uji analisis depskripsi, analisis bivariate menggunakan uji Chi Square, Pearson, dan Spearman.Hasil : Sebanyak 36,1 subjek mengalami stunting. Rerata z-score TB/U -1,25 ± 1,2. Rerata asupan protein, seng, dan zat besi subjek berturut-turut 34.8 ± 13 g, 5.2 ± 2.5 mg, 8.2 ± 6.5 mg dengan sebagian besar tingkat kecukupan protein, seng, dan zat besi subjek adalah cukup. Sebanyak 29.1% subjek memiliki riwayat infeksi. Terdapat hubungan antara protein dan penyakit infeksi dengan z-score TB/U pada Balita. Tidak terdapat hubungan antara asupan seng, dan zat besi dengan z-score TB/U pada Balita. Simpulan : Terdapat hubungan antara asupan protein dan riwayat penyakit infeksi terhadap indeks z-score TB/U pada Balita

Erosion rates in a wet, temperate climate derived from rock luminescence techniques

Abstract. A new luminescence erosion-meter has huge potential for inferring erosion rates on sub-millennial scales for both steady and transient states of erosion, which is currently not possible with any existing techniques capable of measuring erosion. This study applies new rock luminescence techniques to a well-constrained scenario provided by the Beinn Alligin rock avalanche, NW Scotland. Boulders in this deposit are lithologically consistent, have known cosmogenic nuclide ages, and independently-derived Holocene erosion rates. We find that luminescence-derived exposure ages for the Beinn Alligin rock avalanche were an order of magnitude younger than existing cosmogenic nuclide exposure ages, suggestive of high erosion rates (as supported by field evidence of quartz grain protrusions on the rock surfaces). Erosion rates determined by luminescence were consistent with independently-derived rates measured from boulder-edge roundness. Inversion modelling indicates a transient state of erosion reflecting the stochastic nature of erosional processes over the last ~4 ka in the wet, temperate climate of NW Scotland. Erosion was likely modulated by known fluctuations in moisture availability, and to a lesser extent temperature, which controlled the extent of chemical weathering of these highly-lithified rocks prior to erosion. The use of a multi-elevated temperature, post-infra-red, infra-red stimulated luminescence (MET-pIRIR) protocol (50, 150 and 225 °C) was advantageous as it identified samples with complexities introduced by within-sample variability (e.g. surficial coatings). This study demonstrates that the luminescence erosion-meter can infer accurate erosion rates on sub-millennial scales and identify transient states of erosion (i.e. stochastic processes) in agreement with independently-derived erosion rates for the same deposit. </jats:p

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1 -S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1Amutant OCCC. Mol Cancer Ther; 15(7); 1472-84. Ó2016 AACR.</p

Updated threshold dose-distribution data for sesame

Sesame is classified as a “major” food allergen for which mandatory disclosure is required. Understanding reaction thresholds and how these vary within the allergic population is crucial in providing appropriate dietary advice to patients, providing guidance to the food industry, and informing dosing regimens for oral food challenges (FC). However, the largest data series used to derive a threshold dose-distribution for sesame included blinded challenge data from just 40 individuals.1 Data from low-dose, open FC can be used to supplement that from blinded FC, reducing uncertainty in estimating threshold dose-distributions for allergenic foods which otherwise lack sufficient data.2 We, therefore, undertook a systematic search of the literature and performed dose-distribution modelling of individual patient FC data (including open FC) to update estimated eliciting doses for sesame