pLV-SFFV-humanNIS-P2A-eGFP-T2A-Luc2

• Name of plasmid: pLV-SFFV-human NIS-P2A-eGFP-T2A-Luc2
• Description: pHR' SIN-SFFV-hNIS-P2A-eGFP-T2A-Luc2 plasmid DNA
• Amount of DNA: 20 ug
• Promoter: SFFV spleen focus forming virus promoter for high constitutive gene expression in numerous cell types. Unlike the CMV promoter, SFFV works very well in human, rodent, and other animal cells,
• Transgenes: Human sodium iodide symporter (hNIS), enhanced green fluorescent protein (eGFP), and firefly luciferase (Luc2)
• Linkers: Self-cleavage peptides P2A and T2A
• Proposed Use: For generation of lentiviral vector stocks encoding human NIS, enhanced GFP, and firefly luciferase in a triple transfection protocol.

Plasmid Map:

Transgene Validation:

Uptake Assay: NIS mediated ¹²⁵I uptake of LV-hNIS-eGFP-Luc2 transduced cells was assayed in the presence or absence of KClO₄, a competitive inhibitor of iodine uptake.

Flow Cytometry: LV-hNIS-eGFP-Luc2 transduced (green) or control (grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry.

Luciferase Assay: 10⁴, 10⁵, or 10⁶ LV-hNIS-eGFP-Luc2 transduced cells were placed in wells of a 96-well plate and 0.3 mg of d-luciferin was added to the indicated wells. The plate was immediately imaged using a Xenogen IVIS Spectrum.