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One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.
Grav, Lise Marie,Lee, Jae Seong,Gerling, Signe,Kallehauge, Thomas Beuchert,Hansen, Anders Holmgaard,Kol, Stefan,Lee, Gyun Min,Pedersen, Lasse Ebdrup,Kildegaard, Helene Faustrup Wiley 2015 Biotechnology Journal Vol.10 No.9
<P>The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.</P>
OBSERVATIONAL AND DYNAMICAL CHARACTERIZATION OF MAIN-BELT COMET P/2010 R2 (La Sagra)
Hsieh, Henry H.,Yang, Bin,Haghighipour, Nader,Novaković,, Bojan,Jedicke, Robert,Wainscoat, Richard J.,Denneau, Larry,Abe, Shinsuke,Chen, Wen-Ping,Fitzsimmons, Alan,Granvik, Mikael,Grav, Tommy,Ip, American Institute of Physics 2012 The Astronomical journal Vol.143 No.5
<P>We present observations of the recently discovered comet-like main-belt object P/2010 R2 (La Sagra) obtained by Pan-STARRS1 and the Faulkes Telescope-North on Haleakala in Hawaii, the University of Hawaii 2.2 m, Gemini-North, and Keck I telescopes on Mauna Kea, the Danish 1.54 m telescope (operated by the MiNDSTEp consortium) at La Silla, and the Isaac Newton Telescope on La Palma. An antisolar dust tail is observed to be present from 2010 August through 2011 February, while a dust trail aligned with the object's orbit plane is also observed from 2010 December through 2011 August. Assuming typical phase darkening behavior, P/La Sagra is seen to increase in brightness by >1 mag between 2010 August and December, suggesting that dust production is ongoing over this period. These results strongly suggest that the observed activity is cometary in nature (i.e., driven by the sublimation of volatile material), and that P/La Sagra is therefore the most recent main-belt comet to be discovered. We find an approximate absolute magnitude for the nucleus of H<SUB>R</SUB> = 17.9 ± 0.2 mag, corresponding to a nucleus radius of ~0.7 km, assuming an albedo of p = 0.05. Comparing the observed scattering surface areas of the dust coma to that of the nucleus when P/La Sagra was active, we find dust-to-nucleus area ratios of A<SUB>d</SUB>/A<SUB>N</SUB> = 30-60, comparable to those computed for fellow main-belt comets 238P/Read and P/2008 R1 (Garradd), and one to two orders of magnitude larger than for two other main-belt comets (133P/Elst-Pizarro and 176P/LINEAR). Using optical spectroscopy to search for CN emission, we do not detect any conclusive evidence of sublimation products (i.e., gas emission), finding an upper limit CN production rate of Q<SUB>CN</SUB> < 6 × 10<SUP>23</SUP> mol s<SUP>–1</SUP>, from which we infer an H<SUB>2</SUB>O production rate of <img SRC='http://ej.iop.org/images/1538-3881/143/5/104/aj423523ieqn1.gif' ALIGN='MIDDLE' ALT='$Q_{\rm H_2O}\,{<}\,10^{26}$'/> mol s<SUP>–1</SUP>. Numerical simulations indicate that P/La Sagra is dynamically stable for >100 Myr, suggesting that it is likely native to its current location and that its composition is likely representative of other objects in the same region of the main belt, though the relatively close proximity of the 13:6 mean-motion resonance with Jupiter and the (3,–2,–1) three-body mean-motion resonance with Jupiter and Saturn mean that dynamical instability on larger timescales cannot be ruled out.</P>