Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells

Track D Social Science, Human Rights and Political Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138414/1/jia218442.pd

Measurements of differential cross-sections in top-quark pair events with a high transverse momentum top quark and limits on beyond the Standard Model contributions to top-quark pair production with the ATLAS detector at √s = 13 TeV

Cross-section measurements of top-quark pair production where the hadronically decaying top quark has transverse momentum greater than 355 GeV and the other top quark decays into ℓνb are presented using 139 fb−1 of data collected by the ATLAS experiment during proton-proton collisions at the LHC. The fiducial cross-section at s = 13 TeV is measured to be σ = 1.267 ± 0.005 ± 0.053 pb, where the uncertainties reflect the limited number of data events and the systematic uncertainties, giving a total uncertainty of 4.2%. The cross-section is measured differentially as a function of variables characterising the tt¯ system and additional radiation in the events. The results are compared with various Monte Carlo generators, including comparisons where the generators are reweighted to match a parton-level calculation at next-to-next-to-leading order. The reweighting improves the agreement between data and theory. The measured distribution of the top-quark transverse momentum is used to search for new physics in the context of the effective field theory framework. No significant deviation from the Standard Model is observed and limits are set on the Wilson coefficients of the dimension-six operators OtG and Otq(8), where the limits on the latter are the most stringent to date. [Figure not available: see fulltext.]

Direct constraint on the Higgs–charm coupling from a search for Higgs boson decays into charm quarks with the ATLAS detector

A search for the Higgs boson decaying into a pair of charm quarks is presented. The analysis uses proton–proton collisions to target the production of a Higgs boson in association with a leptonically decaying W or Z boson. The dataset delivered by the LHC at a centre-of-mass energy of and recorded by the ATLAS detector corresponds to an integrated luminosity of 139 fb−1. Flavour-tagging algorithms are used to identify jets originating from the hadronisation of charm quarks. The analysis method is validated with the simultaneous measurement of WW, WZ and ZZ production, with observed (expected) significances of 2.6 (2.2) standard deviations above the background-only prediction for the (W/Z)Z(→cc¯) process and 3.8 (4.6) standard deviations for the (W/Z)W(→cq) process. The (W/Z)H(→cc¯) search yields an observed (expected) upper limit of 26 (31) times the predicted Standard Model cross-section times branching fraction for a Higgs boson with a mass of , corresponding to an observed (expected) constraint on the charm Yukawa coupling modifier |κc|<8.5 (12.4), at the 95% confidence level. A combination with the ATLAS (W/Z)H,H→bb¯ analysis is performed, allowing the ratio κc/κb to be constrained to less than 4.5 at the 95% confidence level, smaller than the ratio of the b- and c-quark masses, and therefore determines the Higgs-charm coupling to be weaker than the Higgs-bottom coupling at the 95% confidence level

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Observation of electroweak production of two jets in association with an isolated photon and missing transverse momentum, and search for a Higgs boson decaying into invisible particles at 13 TeV with the ATLAS detector

This paper presents the measurement of the electroweak production of two jets in association with a $Z\gamma$ pair with the $Z$ boson decaying into two neutrinos. It also presents the search for invisible or partially invisible decays of a Higgs boson with a mass of 125 GeV produced through vector-boson fusion with a photon in the final state. These results use data from LHC proton-proton collisions at $\sqrt{s}$ = 13 TeV collected with the ATLAS detector corresponding to an integrated luminosity of 139 fb$^{-1}$. The event signature, shared by all benchmark processes considered for measurements and searches, is characterized by a significant amount of unbalanced transverse momentum and a photon in the final state, in addition to a pair of forward jets. For electroweak production of $Z\gamma$ in association with two jets, the background-only hypothesis is rejected with an observed (expected) significance of 5.2 (5.1) standard deviations. The measured fiducial cross-section for this process is 1.31$\pm$0.29 fb. Observed (expected) upper limit of 0.37 (0.34) at 95% confidence level is set on the branching ratio of a 125 GeV Higgs boson to invisible particles, assuming the Standard Model production cross-section. The signature is also interpreted in the context of decays of a Higgs boson to a photon and a dark photon. An observed (expected) 95% CL upper limit on the branching ratio for this decay is set at 0.018 (0.017), assuming the 125 GeV Standard Model Higgs boson production cross-section