MTC-SK cells were used for cell culture experiments. Those cells were derived from sporadic (non MEN) tumors and do not harbor RET mutations. MTC-SK cells were maintained in culture media containing Ham's F12 (Lonza Group Ltd, Basel, Switzerland), Medium 199 (Sigma, St. Louis, MO, USA) 1:1 v/v and 10% FBS as described in [24 (link)]. For transfections, cells were plated at a density of 2.5x105 cells/ml and transfected with either 1 μg pCMV-Kd-CDK5 [34 (link)] or 1 μg pCMV-EGFP (Clontech Laboratories, Inc., Mountain View, CA, USA) using X-tremeGene HP DNA transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany). Cells were harvested 24 h post-transfection and lysate was prepared as previously described in [42 (link)].
Pcmv egfp
Manufactured by Takara Bio
Sourced in United States
The PCMV-EGFP is a plasmid vector that contains the enhanced green fluorescent protein (EGFP) gene under the control of the cytomegalovirus (CMV) promoter. The EGFP gene serves as a reporter for gene expression studies.
Automatically generated - may contain errors
3 protocols using pcmv egfp
1
Cell Culture and Transfection of MTC-SK Cells
2
Transfection of A549 cells using Lipo2k and BPEI
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The GFP-encoding plasmid DNA (pCMV-EGFP) (Clontech Laboratories Inc., Palo Alto, CA, USA) complex with Lipo2k (Invitrogen) was prepared according to the manufacturer's protocol. BPEI (25 kDa) were prepared at the N/P ratio of 20/1. A549 cells were seeded onto six-well plates at a density of 5×105 cells per well in 2 mL of serum-free media and grown to reach 60-80% confluence. The culture medium was replaced with 2 mL of the transfection medium containing the Lipo2k or BPEI complex, followed by 6 h incubation at 37 °C. Then, the transfection medium was replaced with fresh complete RPMI 1640 (10% FBS), and the cells were allowed to grow for 48 h. After incubation, the cells were washed twice with DPBS (pH 7.4), and flow cytometry analysis was performed by the same method as described above.
3
SB100X Transposon Plasmid Construction
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SB100X transposase variant was expressed from a standard expression plasmid regulated by a CMV promoter (pCMV-SB100X) (Mates et al., 2009) . SB transposon donor plasmids were created from the pT2 variant of the SB inverted repeat-direct repeats (IRDRs) (Geurts et al., 2003) . Puromycin antibiotic resistance gene was cloned into the initial transposon plasmid with PCR technique; CAG, CMV, PGK, and SV40 promoters were cloned with standard restriction cassette replacements. For GFP expression plasmids, the puromycin coding sequences in these transposon donor plasmids were changed to GFP using restriction enzymes. For transfection control, the pCMV-EGFP (Clontech) expression plasmid was used.
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