122 research outputs found
Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector
BACKGROUND: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers.
METHODS: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells.
RESULTS: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs.
CONCLUSION: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications
Longitudinal double-spin asymmetry and cross section for inclusive neutral pion production at midrapidity in polarized proton collisions at sqrt(s) = 200 GeV
We report a measurement of the longitudinal double-spin asymmetry A_LL and
the differential cross section for inclusive Pi0 production at midrapidity in
polarized proton collisions at sqrt(s) = 200 GeV. The cross section was
measured over a transverse momentum range of 1 < p_T < 17 GeV/c and found to be
in good agreement with a next-to-leading order perturbative QCD calculation.
The longitudinal double-spin asymmetry was measured in the range of 3.7 < p_T <
11 GeV/c and excludes a maximal positive gluon polarization in the proton. The
mean transverse momentum fraction of Pi0's in their parent jets was found to be
around 0.7 for electromagnetically triggered events.Comment: 6 pages, 3 figures, submitted to Phys. Rev. D (RC
Detector Description and Performance for the First Coincidence Observations between LIGO and GEO
For 17 days in August and September 2002, the LIGO and GEO interferometer
gravitational wave detectors were operated in coincidence to produce their
first data for scientific analysis. Although the detectors were still far from
their design sensitivity levels, the data can be used to place better upper
limits on the flux of gravitational waves incident on the earth than previous
direct measurements. This paper describes the instruments and the data in some
detail, as a companion to analysis papers based on the first data.Comment: 41 pages, 9 figures 17 Sept 03: author list amended, minor editorial
change
Search for Gravitational Waves from Primordial Black Hole Binary Coalescences in the Galactic Halo
We use data from the second science run of the LIGO gravitational-wave
detectors to search for the gravitational waves from primordial black hole
(PBH) binary coalescence with component masses in the range 0.2--.
The analysis requires a signal to be found in the data from both LIGO
observatories, according to a set of coincidence criteria. No inspiral signals
were found. Assuming a spherical halo with core radius 5 kpc extending to 50
kpc containing non-spinning black holes with masses in the range 0.2--, we place an observational upper limit on the rate of PBH coalescence
of 63 per year per Milky Way halo (MWH) with 90% confidence.Comment: 7 pages, 4 figures, to be submitted to Phys. Rev.
Longitudinal scaling property of the charge balance function in Au + Au collisions at 200 GeV
We present measurements of the charge balance function, from the charged
particles, for diverse pseudorapidity and transverse momentum ranges in Au + Au
collisions at 200 GeV using the STAR detector at RHIC. We observe that the
balance function is boost-invariant within the pseudorapidity coverage [-1.3,
1.3]. The balance function properly scaled by the width of the observed
pseudorapidity window does not depend on the position or size of the
pseudorapidity window. This scaling property also holds for particles in
different transverse momentum ranges. In addition, we find that the width of
the balance function decreases monotonically with increasing transverse
momentum for all centrality classes.Comment: 6 pages, 3 figure
Measurement of the Bottom contribution to non-photonic electron production in collisions at =200 GeV
The contribution of meson decays to non-photonic electrons, which are
mainly produced by the semi-leptonic decays of heavy flavor mesons, in
collisions at 200 GeV has been measured using azimuthal
correlations between non-photonic electrons and hadrons. The extracted
decay contribution is approximately 50% at a transverse momentum of GeV/. These measurements constrain the nuclear modification factor for
electrons from and meson decays. The result indicates that meson
production in heavy ion collisions is also suppressed at high .Comment: 6 pages, 4 figures, accepted by PR
Isolation of chromosome clusters from metaphase-arrested HeLa cells
We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosomes from a cell remain together in a bundle which we call a “metaphase chromosome cluster”. Cells are arrested to 90–95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a Percoll TM density gradient. — This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca ++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47359/1/412_2004_Article_BF00327351.pd
System size dependence of associated yields in hadron-triggered jets
We present results on the system size dependence of high transverse momentum
di-hadron correlations at = 200 GeV as measured by STAR at
RHIC. Measurements in d+Au, Cu+Cu and Au+Au collisions reveal similar jet-like
correlation yields at small angular separation (,
) for all systems and centralities. Previous measurements have
shown that the away-side yield is suppressed in heavy-ion collisions. We
present measurements of the away-side suppression as a function of transverse
momentum and centrality in Cu+Cu and Au+Au collisions. The suppression is found
to be similar in Cu+Cu and Au+Au collisions at a similar number of
participants. The results are compared to theoretical calculations based on the
parton quenching model and the modified fragmentation model. The observed
differences between data and theory indicate that the correlated yields
presented here will provide important constraints on medium density profile and
energy loss model parameters.Comment: 12 pages, 5 figure
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