High-titer purified LV-hNIS-P2A-eGFP-T2A-Luc2 lentivirus stocks

Promoter: Spleen focus-forming virus (SFFV) promoter for high constitutive gene expression in numerous cell types. Unlike the CMV promoter, we find that SFFV works very well in animal models.

Reporter genes: Human sodium iodide symporter (hNIS), Enhanced green fluorescent protein (eGFP), Firefly luciferase (Luc2).

Envelope/Tropism: VSV.G pseudotyped HIV-1 based lentiviral vector capable of transducing a broad range of cell types and species.

Description: This is a ready-to-use lentivirus preparation. The virus encodes the human sodium iodide symporter (hNIS) cDNA under control of the spleen focus-forming virus (SFFV) promoter linked to the enhanced green fluorescent protein (eGFP) cDNA via a P2A cleavage peptide and the firefly luciferase (Luc2) cDNA via a T2A cleavage peptide (see below). This is a self-inactivating (SIN) vector in which the viral enhancer and promoter have been deleted. Thus, transcription inactivation of the LTR in the SIN provirus increases biosafety by preventing mobilization of replication competent viruses and enables regulated expression of the genes from the internal promoters without cis-acting effects of the LTR (Miyoshi et al., J Virol. 1998).

Titer: Approximately 5e7 TU/mL (by qPCR titration of transduced HeLa H1 cells)

Proposed Use: In vitro transduction of primary cells and cell lines.

Vector Map:

 

Iodine (I-125) Uptake Assay of LV-hNIS-P2A-eGFP-T2A-Luc2 transduced HeLa H1 cells: HeLaH1 cells were transduced with LV-hNIS-P2A-eGFP-T2A-Luc2 at the indicated MOIs. After 1 week I-125 uptake by the cells was measured in the presence or absence of KClO₄, an inhibitor of NIS-mediated I-125 uptake.

Fluorescence Expression: HeLaH1 cells were transduced with LV-hNIS-P2A-eGFP-T2A-Luc2 at the indicated MOIs. After 6 days fluorescence expression of the transduced (green) and untransducted parental HeLaH1 cells (grey) was analyzed by flow cytometry.

Luciferase Expression: HeLaH1 cells were transduced with LV-hNIS-P2A-eGFP-T2A-Luc2 at the indicated MOIs. To measure luciferase expression, 10⁴, 10⁵, or 10⁶ cells were seeded in wells of a 96-well plate and 3 mg/mL of d-luciferin was added to the indicated wells. The plate was immediately imaged using the IVIS Spectrum imaging system.