Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines

Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out

Study of electron anti-neutrinos associated with gamma-ray bursts using KamLAND

We search for electron anti-neutrinos ($\overline{\nu}_e$) from long and short-duration gamma-ray bursts~(GRBs) using data taken by the KamLAND detector from August 2002 to June 2013. No statistically significant excess over the background level is found. We place the tightest upper limits on $\overline{\nu}_e$ fluence from GRBs below 7 MeV and place first constraints on the relation between $\overline{\nu}_e$ luminosity and effective temperature.Comment: 16 pages and 5 figure

White paper: CeLAND - Investigation of the reactor antineutrino anomaly with an intense 144Ce-144Pr antineutrino source in KamLAND

We propose to test for short baseline neutrino oscillations, implied by the recent reevaluation of the reactor antineutrino flux and by anomalous results from the gallium solar neutrino detectors. The test will consist of producing a 75 kCi 144Ce - 144Pr antineutrino source to be deployed in the Kamioka Liquid Scintillator Anti-Neutrino Detector (KamLAND). KamLAND's 13m diameter target volume provides a suitable environment to measure energy and position dependence of the detected neutrino flux. A characteristic oscillation pattern would be visible for a baseline of about 10 m or less, providing a very clean signal of neutrino disappearance into a yet-unknown, "sterile" state. Such a measurement will be free of any reactor-related uncertainties. After 1.5 years of data taking the Reactor Antineutrino Anomaly parameter space will be tested at > 95% C.L.Comment: White paper prepared for Snowmass-2013; slightly different author lis

KamLAND Sensitivity to Neutrinos from Pre-Supernova Stars

In the late stages of nuclear burning for massive stars (M>8~M_{\sun}), the production of neutrino-antineutrino pairs through various processes becomes the dominant stellar cooling mechanism. As the star evolves, the energy of these neutrinos increases and in the days preceding the supernova a significant fraction of emitted electron anti-neutrinos exceeds the energy threshold for inverse beta decay on free hydrogen. This is the golden channel for liquid scintillator detectors because the coincidence signature allows for significant reductions in background signals. We find that the kiloton-scale liquid scintillator detector KamLAND can detect these pre-supernova neutrinos from a star with a mass of 25~M_{\sun} at a distance less than 690~pc with 3$\sigma$ significance before the supernova. This limit is dependent on the neutrino mass ordering and background levels. KamLAND takes data continuously and can provide a supernova alert to the community.Comment: 19 pages, 6 figures, 1 tabl

A compact ultra-clean system for deploying radioactive sources inside the KamLAND detector

We describe a compact, ultra-clean device used to deploy radioactive sources along the vertical axis of the KamLAND liquid-scintillator neutrino detector for purposes of calibration. The device worked by paying out and reeling in precise lengths of a hanging, small-gauge wire rope (cable); an assortment of interchangeable radioactive sources could be attached to a weight at the end of the cable. All components exposed to the radiopure liquid scintillator were made of chemically compatible UHV-cleaned materials, primarily stainless steel, in order to avoid contaminating or degrading the scintillator. To prevent radon intrusion, the apparatus was enclosed in a hermetically sealed housing inside a glove box, and both volumes were regularly flushed with purified nitrogen gas. An infrared camera attached to the side of the housing permitted real-time visual monitoring of the cable's motion, and the system was controlled via a graphical user interface.Comment: Revised author affiliations, corrected typos, made minor improvements to text, and revised reference

Search for extraterrestrial antineutrino sources with the KamLAND detector

We present the results of a search for extraterrestrial electron antineutrinos ($\bar{\nu}_{e}$'s) in the energy range $8.3 MeV < E_{\bar{\nu}_{e}} < 31.8 MeV$ using the KamLAND detector. In an exposure of 4.53 kton-year, we identify 25 candidate events. All of the candidate events can be attributed to background, most importantly neutral current atmospheric neutrino interactions, setting an upper limit on the probability of $^{8}$B solar $\nu_{e}$'s converting into $\bar{\nu}_{e}$'s at $5.3 \times 10^{-5}$ (90% C.L.), if we assume an undistorted $\bar{\nu}_{e}$ shape. This limit corresponds to a solar $\bar{\nu}_{e}$ flux of $93 cm^{-2} s^{-1}$ or an event rate of $1.6 events (kton-year)^{-1}$ above the energy threshold $(E_{\bar{\nu}_{e}} > 8.3 MeV)$. The present data also allows us to set more stringent limits on the diffuse supernova neutrino flux and on the annihilation rates for light dark matter particles.Comment: 22 pages, 6 figure

Production of Radioactive Isotopes through Cosmic Muon Spallation in KamLAND

Radioactive isotopes produced through cosmic muon spallation are a background for rare-event detection in $\nu$ detectors, double-$\beta$-decay experiments, and dark-matter searches. Understanding the nature of cosmogenic backgrounds is particularly important for future experiments aiming to determine the pep and CNO solar neutrino fluxes, for which the background is dominated by the spallation production of $^{11}$C. Data from the Kamioka liquid-scintillator antineutrino detector (KamLAND) provides valuable information for better understanding these backgrounds, especially in liquid scintillators, and for checking estimates from current simulations based upon MUSIC, FLUKA, and GEANT4. Using the time correlation between detected muons and neutron captures, the neutron production yield in the KamLAND liquid scintillator is measured to be $(2.8 \pm 0.3) \times 10^{-4} \mu^{-1} g^{-1} cm^{2}$. For other isotopes, the production yield is determined from the observed time correlation related to known isotope lifetimes. We find some yields are inconsistent with extrapolations based on an accelerator muon beam experiment.Comment: 16 pages, 20 figure

Measurement of the 8B Solar Neutrino Flux with the KamLAND Liquid Scintillator Detector

We report a measurement of the neutrino-electron elastic scattering rate from 8B solar neutrinos based on a 123 kton-day exposure of KamLAND. The background-subtracted electron recoil rate, above a 5.5 MeV analysis threshold is 1.49+/-0.14(stat)+/-0.17(syst) events per kton-day. Interpreted as due to a pure electron flavor flux with a 8B neutrino spectrum, this corresponds to a spectrum integrated flux of 2.77+/-0.26(stat)+/-0.32(syst) x 10^6 cm^-2s^-1. The analysis threshold is driven by 208Tl present in the liquid scintillator, and the main source of systematic uncertainty is due to background from cosmogenic 11Be. The measured rate is consistent with existing measurements and with Standard Solar Model predictions which include matter enhanced neutrino oscillation.Comment: 6 pages, 3 figure