Efficient, high-throughput transfection of human embryonic stem cells

INTRODUCTION: Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. METHODS: We investigated the ability of two commercially available electroporation systems, the Nucleofection(® )96-well Shuttle(® )System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. RESULTS: Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection(® )96-well Shuttle(® )System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. CONCLUSIONS: Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC

A non-transformed oligodendrocyte precursor cell line, OL-1, facilitates studies of insulin-like growth factor-I signaling during oligodendrocyte development

The process by which oligodendrocyte progenitors differentiate into mature oligodendrocytes is complex and incompletely understood in part because of the paucity of oligodendrocyte precursors cell lines that can be studied in culture. We have developed a non-immortalized rat oligodendrocyte precursor line, called OL-1, which behaves in a fashion consistent with developing oligodendrocytes in vivo. This OL-1 line provides a model for the study of oligodendrocyte development and offers an alternative to the CG-4 cell line. When OL-1 cells are propagated in conditioned growth media, they have morphology consistent with immature oligodendrocytes and exhibit A2B5 antigen positive and myelin basic protein-negative immunoreactivity. Withdrawal of conditioned growth media and culture in serum-free medium results in OL-1 cell maturation, manifested by a shift to myelin basic protein-positive immunoreactivity, A2B5 antigen-negative immunoreactivity, decreased NG2 mRNA expression, increased expression of proteolipid protein mRNA, and increased expression of CNP protein. In addition, the expression of proteolipid protein and its splicing variant DM-20 exhibit a pattern that is similar to brain proteolipid protein expression during development. When OL-1 cells are exposed to Insulin-like growth factor-I, there are significant increases in proteolipid protein mRNA expression ( p < 0.05), the number of cell processes ( p < 0.05), and cell number ( p < 0.05). Treatment with the caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK (inhibitors of caspases 3, 6, 7, 8, 10 and 1, 3, 4, respectively), Insulin-like growth factor-I, or both, results in a similar increase in cell number. Because Insulin-like growth factor-I does not substantially increase the BrdU labeling of OL-1 cells, these data collectively indicate that Insulin-like growth factor-I increases OL-1 cell number predominately by promoting survival, rather than stimulating proliferation. This non-immortalized oligodendrocyte precursor cell line, therefore, exhibits behavior consistent with the in vivo development of oligodendrocytes and provides an excellent model for the study of developing oligodendrocytes

Reverberation Mapping of the Kepler-Field AGN KA1858+4850

KA1858+4850 is a narrow-line Seyfert 1 galaxy at redshift 0.078 and is among the brightest active galaxies monitored by the Kepler mission. We have carried out a reverberation mapping campaign designed to measure the broad-line region size and estimate the mass of the black hole in this galaxy. We obtained 74 epochs of spectroscopic data using the Kast Spectrograph at the Lick 3-m telescope from February to November of 2012, and obtained complementary V-band images from five other ground-based telescopes. We measured the H-beta light curve lag with respect to the V-band continuum light curve using both cross-correlation techniques (CCF) and continuum light curve variability modeling with the JAVELIN method, and found rest-frame lags of lag_CCF = 13.53 (+2.03, -2.32) days and lag_JAVELIN = 13.15 (+1.08, -1.00) days. The H-beta root-mean-square line profile has a width of sigma_line = 770 +/- 49 km/s. Combining these two results and assuming a virial scale factor of f = 5.13, we obtained a virial estimate of M_BH = 8.06 (+1.59, -1.72) x 10^6 M_sun for the mass of the central black hole and an Eddington ratio of L/L_Edd ~ 0.2. We also obtained consistent but slightly shorter emission-line lags with respect to the Kepler light curve. Thanks to the Kepler mission, the light curve of KA1858+4850 has among the highest cadences and signal-to-noise ratios ever measured for an active galactic nucleus; thus, our black hole mass measurement will serve as a reference point for relations between black hole mass and continuum variability characteristics in active galactic nuclei

Search for Yukawa Production of a Light Neutral Higgs Boson at LEP

Within a Two-Higgs-Doublet Model (2HDM) a search for a light Higgs boson in the mass range of 4-12 GeV has been performed in the Yukawa process e+e- -> b bbar A/h -> b bbar tau+tau-, using the data collected by the OPAL detector at LEP between 1992 and 1995 in e+e- collisions at about 91 GeV centre-of-mass energy. A likelihood selection is applied to separate background and signal. The number of observed events is in good agreement with the expected background. Within a CP-conserving 2HDM type II model the cross-section for Yukawa production depends on xiAd = |tan beta| and xihd = |sin alpha/cos beta| for the production of the CP-odd A and the CP-even h, respectively, where tan beta is the ratio of the vacuum expectation values of the Higgs doublets and alpha is the mixing angle between the neutral CP-even Higgs bosons. From our data 95% C.L. upper limits are derived for xiAd within the range of 8.5 to 13.6 and for xihd between 8.2 to 13.7, depending on the mass of the Higgs boson, assuming a branching fraction into tau+tau- of 100%. An interpretation of the limits within a 2HDM type II model with Standard Model particle content is given. These results impose constraints on several models that have been proposed to explain the recent BNL measurement of the muon anomalous magnetic moment.Comment: 24 pages, 9 figures, Submitted to Euro. Phys. J.

Measurement of triple gauge boson couplings from WW production at LEP energies up to 189 GeV

A measurement of triple gauge boson couplings is presented, based on W-pair data recorded by the OPAL detector at LEP during 1998 at a centre-of-mass energy of 189 GeV with an integrated luminosity of 183 pb^-1. After combining with our previous measurements at centre-of-mass energies of 161-183 GeV we obtain k_g=0.97 +0.20 -0.16, g_1^z=0.991 +0.060 -0.057 and lambda_g=-0.110 +0.058 -0.055, where the errors include both statistical and systematic uncertainties and each coupling is determined by setting the other two couplings to their SM values. These results are consistent with the Standard Model expectations.Comment: 28 pages, 8 figures, submitted to Eur. Phys. J.

Measurement of the Hadronic Cross-Section for the Scattering of Two Virtual Photons at LEP

The interaction of virtual photons is investigated using the reaction e+e- -> e+e- hadrons based on data taken by the OPAL experiment at e+e- centre-of-mass energies sqrt(s_ee)=189-209 GeV, for W>5 GeV and at an average Q^2 of 17.9 GeV^2. The measured cross-sections are compared to predictions of the Quark Parton Model (QPM), to the Leading Order QCD Monte Carlo model PHOJET to the NLO prediction for the reaction e+e- -> e+e-qqbar, and to BFKL calculations. PHOJET, NLO e+e- -> e+e-qqbar, and QPM describe the data reasonably well, whereas the cross-section predicted by a Leading Order BFKL calculation is too large.Comment: 30 pages, 10 figures, Submitted to Eur.Phys.J.

Search for Neutral Higgs Bosons in e+e- Collisions at sqrt(s) ~189GeV

A search for neutral Higgs bosons has been performed with the OPAL detector at LEP, using approximately 170 pb-1 of e+e- collision data collected at sqrt(s)~189GeV. Searches have been performed for the Standard Model (SM) process e+e- to H0Z0 and the MSSM processes e+e- to H0Z0, A0h0. The searches are sensitive to the b b-bar and tau antitau decay modes of the Higgs bosons, and also to the MSSM decay mode h0 to A0A0. OPAL search results at lower centre-of-mass energies have been incorporated in the limits we set, which are valid at the 95% confidence level. For the SM Higgs boson, we obtain a lower mass bound of 91.0 GeV. In the MSSM, our limits are mh>74.8GeV and mA>76.5GeV, assuming tan(beta)>1, that the mixing of the scalar top quarks is either zero or maximal, and that the soft SUSY-breaking masses are 1 TeV. For the case of zero scalar top mixing, we exclude values of tan(beta) between 0.72 and 2.19.Comment: 38 pages, 15 figures, submitted Euro. Phys. J.

Search for Higgs Bosons in e+e- Collisions at 183 GeV

The data collected by the OPAL experiment at sqrts=183 GeV were used to search for Higgs bosons which are predicted by the Standard Model and various extensions, such as general models with two Higgs field doublets and the Minimal Supersymmetric Standard Model (MSSM). The data correspond to an integrated luminosity of approximately 54pb-1. None of the searches for neutral and charged Higgs bosons have revealed an excess of events beyond the expected background. This negative outcome, in combination with similar results from searches at lower energies, leads to new limits for the Higgs boson masses and other model parameters. In particular, the 95% confidence level lower limit for the mass of the Standard Model Higgs boson is 88.3 GeV. Charged Higgs bosons can be excluded for masses up to 59.5 GeV. In the MSSM, mh > 70.5 GeV and mA > 72.0 GeV are obtained for tan{beta}>1, no and maximal scalar top mixing and soft SUSY-breaking masses of 1 TeV. The range 0.8 < tanb < 1.9 is excluded for minimal scalar top mixing and m{top} < 175 GeV. More general scans of the MSSM parameter space are also considered.Comment: 49 pages. LaTeX, including 33 eps figures, submitted to European Physical Journal

A Measurement of the Product Branching Ratio f(b->Lambda_b).BR(Lambda_b->Lambda X) in Z0 Decays

The product branching ratio, f(b->Lambda_b).BR(Lambda_b->Lambda X), where Lambda_b denotes any weakly-decaying b-baryon, has been measured using the OPAL detector at LEP. Lambda_b are selected by the presence of energetic Lambda particles in bottom events tagged by the presence of displaced secondary vertices. A fit to the momenta of the Lambda particles separates signal from B meson and fragmentation backgrounds. The measured product branching ratio is f(b->Lambda_b).BR(Lambda_b->Lambda X) = (2.67+-0.38(stat)+0.67-0.60(sys))% Combined with a previous OPAL measurement, one obtains f(b->Lambda_b).BR(Lambda_b->Lambda X) = (3.50+-0.32(stat)+-0.35(sys))%.Comment: 16 pages, LaTeX, 3 eps figs included, submitted to the European Physical Journal