Mild and transient heat shock enhances DNA integration following lipofection of recombinant plasmids in 4T1 cells

316-320Cancer cells having stably integrated genes encoding tumor-associated antigens could be utilized as a vaccine, in-vitro stimulators of antigen-primed T-cells, and target for cytotoxicity assay, etc. Lipofection is a simple and safer technique for stable transfection of plasmid DNA. However, the poor rate of genomic integration has limited its application. In the current study, the effect of mild and transient heat shock following lipofection on the improvement of genomic integration was evaluated. The cDNA fragments encoding chicken MMP-11peptide (V32-K365) and the immunoglobulin-like domain 2 of chicken VEGFR-2 were cloned separately into pcDNA3.1 vector. Lipofection was carried out using Lipofectamine® 2000 (Life Technologies, USA) in 4T1 cells followed by a heat shock at 42°C for 10 min. Transfected cells were selected for a period of four weeks against 500 µg/mL G418 in RPMI 1640 media supplemented with 10% fetal bovine serum. Distinct G418-resistant colonies appeared after 14 days of selection. Heat shock significantly (P <0.05) increased the number of viable colonies following antibiotic selection. The immunofluorescent study confirmed the stable integration of the target DNAs into the cells. It is concluded that mild and brief heat shock following lipofection improves the stable integration of recombinant pcDNA3.1 plasmids into 4T1 cells

Mild and transient heat shock enhances DNA integration following lipofection of recombinant plasmids in 4T1 cells

Cancer cells having stably integrated genes encoding tumor-associated antigens could be utilized as a vaccine, in-vitro stimulators of antigen-primed T-cells, and target for cytotoxicity assay, etc. Lipofection is a simple and safer technique for stable transfection of plasmid DNA. However, the poor rate of genomic integration has limited its application. In the current study, the effect of mild and transient heat shock following lipofection on the improvement of genomic integration was evaluated. The cDNA fragments encoding chicken MMP-11peptide (V32-K365) and the immunoglobulin-like domain 2 of chicken VEGFR-2 were cloned separately into pcDNA3.1 vector. Lipofection was carried out using Lipofectamine® 2000 (Life Technologies, USA) in 4T1 cells followed by a heat shock at 42°C for 10 min. Transfected cells were selected for a period of four weeks against 500 µg/mL G418 in RPMI 1640 media supplemented with 10% fetal bovine serum. Distinct G418-resistant colonies appeared after 14 days of selection. Heat shock significantly (P &lt;0.05) increased the number of viable colonies following antibiotic selection. The immunofluorescent study confirmed the stable integration of the target DNAs into the cells. It is concluded that mild and brief heat shock following lipofection improves the stable integration of recombinant pcDNA3.1 plasmids into 4T1 cells

Search for heavy resonances in the W/Z-tagged dijet mass spectrum in pp collisions at 7 TeV

A search has been made for massive resonances decaying into a quark and a vector boson, qW or qZ, or a pair of vector bosons, WW, WZ, or ZZ, where each vector boson decays to hadronic final states. This search is based on a data sample corresponding to an integrated luminosity of 5.0 fb 121 of proton\u2013proton collisions collected in the CMS experiment at the LHC in 2011 at a center-of-mass energy of 7 TeV. For sufficiently heavy resonances the decay products of each vector boson are merged into a single jet, and the event effectively has a dijet topology. The background from QCD dijet events is reduced using recently developed techniques that resolve jet substructure. A 95% CL lower limit is set on the mass of excited quark resonances decaying into qW (qZ) at 2.38 TeV (2.15 TeV) and upper limits are set on the cross section for resonances decaying to qW, qZ, WW, WZ, or ZZ final states

Search for long-lived particles in events with photons and missing energy in proton\u2013proton collisions at sqrt(s)=7 TeV

Results are presented from a search for long-lived neutralinos decaying into a photon and an invisible particle, a signature associated with gauge-mediated supersymmetry breaking in supersymmetric models. The analysis is based on a 4.9 inverse femtobarn sample of proton-proton collisions at 1as = 7 TeV, collected with the CMS detector at the LHC. The missing transverse energy and the time of arrival of the photon at the electromagnetic calorimeter are used to search for an excess of events over the expected background. No significant excess is observed, and lower limits at the 95% confidence level are obtained on the mass of the lightest neutralino, m(neutralino) > 220 GeV (for c tau 6000 mm (for m(neutralino) < 150 GeV)

J Exp Med

To investigate the role of Lyt-2 and Thy-1 in cytolysis, we have generated, by ethyl methanesulfonate mutagenesis and selection, variants of the cloned cytolytic T lymphocyte line L3 that specifically lack either Lyt-2 or Thy-1. An analysis of these variants indicates that neither Lyt-2 nor Lyt-3 is responsible for the lethal hit, but suggests that Lyt-2 and/or Lyt-3 are required for an antigen receptor functional in cytolysis. The data also suggest that the expression of Lyt-3 on the cell surface is not independent of the expression of Lyt-2. Finally the data indicate the Thy-1 plays no role in cytolysis.6788878PMC218609