Pcdna3.1 myc his lacz

C2C12 (ATCC, LGC Standards GmbH, Wesel, Germany, catalogue number CRL-1772) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum. Plasmid DNAs were transfected into 50% confluent cells using PromoFectin (PromoKine, Heidelberg, Germany). For luciferase assays, cells were co-transfected with the pGL3-TATA-Desmef3 reporter gene together with the β-galactosidase expression vector pcDNA3.1/Myc-His(+)/lacZ (Invitrogen GmbH, Karlsruhe, Germany) to account for possible differences in transfection efficiency. Cells were harvested 24 h post transfection and firefly luciferase activities were normalised to β-galactosidase activities within the same samples.
Synthetic siRNA duplexes were purchased from Eurofins MWG Operon (Ebersberg Germany). C2C12 cells were transfected by nucleofection with an siRNA targeting the mouse TRIP6 mRNA sequence GUCUGGAUGCUGAGAUAGA28 (link) or a control siRNA targeting dsRed60 (link) using the Cell Line Nucleofector^® Kit V (Lonza, Cologne, Germany). Cells were then seeded at low cell density to allow proliferation.

S2R + cells were transfected with 1 μg of the appropriate plasmid (either pGL3B-Ba1LTIR, pGL3B-Ba3LTIR pGL3B-copia or the empty pGL3B). Renilla luciferase construct (pRL-SV40; Promega, Madison, WI, USA) was used for normalization. Luciferase expression was measured by the detection of luminescence using the dual luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer instructions. Measurements were recorded on GLOMAX 20/20 luminometer (Promega, Madison, WI, USA). The average expression level from three replicate transfections was normalized to the Renilla luciferase co-transfection control. This value was further normalized to the average expression level from three normalized replicates of the pGL3B-copia plasmid to yield a relative luciferase activity estimate.
For the luciferase activity suppression assay HeLa cells were previously transfected with plasmid expressing either transposase (pcDNA/ASE3, pcDNA/ASE1) or β-Galactosidase (pcDNA3.1/myc-His(−)/lacZ) (Invitrogen, Carlsbad, CA, USA).
Error bars represent the standard deviation. Student’s t test was used to evaluate statistical significance.

Genes encoding polyepitope immunogens TCI-N, TCI-N2, and TCI-N3 were cloned into the pcDNA3.1/Myc-His(+)/lacZ (Invitrogen, USA) plasmid vector. As a result three target plasmids P1, P2, and P3 were obtained. Sequences of cloned genes and plasmid fragments containing the promoter region were verified by sequencing.