High-titer purified LV-hNISplus-PGK-Puro 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. Phosphoglycerate kinase promoter for lower level constitutive expression in numerous cell types.

Reporter gene: New human sodium iodide symporter (hNIS) with enhanced uptake of the PET tracer, F18-tetrafluoroborate 

Selection gene: Puromycin (Puro)

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 NIS genetically modified for enhanced uptake for tetrafluoroborate (NISplus). The hNISplus cDNA is under control of the spleen focus-forming virus (SFFV) promoter and the puromycin resistance gene (Puro) under the phosphoglycerate kinase (PGK) promoter (see below). The hNIS also encodes a HA tag which allows easy detection of hNIS expression using anti-HA antibodies. The lentiviral vectors are self-inactivating (SIN) vectors in which the viral enhancer and promoter have been deleted. Transcription inactivation of the LTR in the SIN provirus increases biosafety by preventing mobilization by 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 0.5-2e7 TU/mL (by qPCR titration of transduced HeLa H1 cells)

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

 

 Vector Map:

 

NIS Expression: The functionality of the hNIS gene was assessed using an uptake assay; uptake of F18-tetrafluoroborate (F18-TFB) by lentivirus-transduced HeLaH1 cells in 96-well plates was measured in the presence or absence of KClO₄, an inhibitor of NIS-mediated F18-TFB uptake. Optimized hNISplus increases uptake by 30% as compared to WT hNIS.