PMC:2518050 / 7239-8528 JSONTXT

{"project":"0_colil","denotations":[{"id":"18982121-12357963-359864","span":{"begin":94,"end":98},"obj":"12357963"}],"text":"Ear fibroblasts were prepared from positive TgH1aV founders as described (Schonig and Bujard, 2003). Cells were trypsinized after reaching confluency and plated into 6-well plates divided into sets without and with doxycycline (dox, 1 g/L) (4-[Dimethlamino]-1, 4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide; Sigma–Aldrich, St. Louis, MO). After reaching ∼50% confluency, both the dox+ and the dox− cultures were transfected with 0.5 mg of synthetic reverse tTA (rtTA-M2s) and 0.5 mg of RL plasmids (Promega, Mannheim, Germany) using lipofectamine-2000 DNA transfection reagents as recommended by the vendor (Invitrogen Life Technologies, Carlsbad, CA). After 48 hours, cells were washed once with phosphate-buffered saline (PBS) and incubated in 0.5 ml of lysis buffer on ice. A 50 ml aliquot from each lysate was tested for FL and RL activity (Lumat LB 9581; Berthold Technologies, Wildbad, Germany). The ratio of FL to RL activity was used to correct for DNA transfection efficacy. Individual transfections and measurements were done in duplicate, usually resulting in normalized activity values that agreed within 5%. The founder showing the highest transactivation potential on ear fibroblasts (#14) was used for further analysis."}

{"project":"TEST0","denotations":[{"id":"18982121-94-102-359864","span":{"begin":94,"end":98},"obj":"[\"12357963\"]"}],"text":"Ear fibroblasts were prepared from positive TgH1aV founders as described (Schonig and Bujard, 2003). Cells were trypsinized after reaching confluency and plated into 6-well plates divided into sets without and with doxycycline (dox, 1 g/L) (4-[Dimethlamino]-1, 4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide; Sigma–Aldrich, St. Louis, MO). After reaching ∼50% confluency, both the dox+ and the dox− cultures were transfected with 0.5 mg of synthetic reverse tTA (rtTA-M2s) and 0.5 mg of RL plasmids (Promega, Mannheim, Germany) using lipofectamine-2000 DNA transfection reagents as recommended by the vendor (Invitrogen Life Technologies, Carlsbad, CA). After 48 hours, cells were washed once with phosphate-buffered saline (PBS) and incubated in 0.5 ml of lysis buffer on ice. A 50 ml aliquot from each lysate was tested for FL and RL activity (Lumat LB 9581; Berthold Technologies, Wildbad, Germany). The ratio of FL to RL activity was used to correct for DNA transfection efficacy. Individual transfections and measurements were done in duplicate, usually resulting in normalized activity values that agreed within 5%. The founder showing the highest transactivation potential on ear fibroblasts (#14) was used for further analysis."}

{"project":"2_test","denotations":[{"id":"18982121-12357963-38286609","span":{"begin":94,"end":98},"obj":"12357963"}],"text":"Ear fibroblasts were prepared from positive TgH1aV founders as described (Schonig and Bujard, 2003). Cells were trypsinized after reaching confluency and plated into 6-well plates divided into sets without and with doxycycline (dox, 1 g/L) (4-[Dimethlamino]-1, 4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide; Sigma–Aldrich, St. Louis, MO). After reaching ∼50% confluency, both the dox+ and the dox− cultures were transfected with 0.5 mg of synthetic reverse tTA (rtTA-M2s) and 0.5 mg of RL plasmids (Promega, Mannheim, Germany) using lipofectamine-2000 DNA transfection reagents as recommended by the vendor (Invitrogen Life Technologies, Carlsbad, CA). After 48 hours, cells were washed once with phosphate-buffered saline (PBS) and incubated in 0.5 ml of lysis buffer on ice. A 50 ml aliquot from each lysate was tested for FL and RL activity (Lumat LB 9581; Berthold Technologies, Wildbad, Germany). The ratio of FL to RL activity was used to correct for DNA transfection efficacy. Individual transfections and measurements were done in duplicate, usually resulting in normalized activity values that agreed within 5%. The founder showing the highest transactivation potential on ear fibroblasts (#14) was used for further analysis."}