High-titer purified LV-hTyrosinase-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: Human sodium iodide symporter (hNIS) 

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 tyrosinase (hTyr) cDNA 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 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:

Cell photos of (A) parental and (B) LV-hTyrosinase-PGK-Puro transduced A549 cells: A549 cells were transduced with LV-hTyrosinase-PGK-Puro (MOI = 10). Photos of the parental cells (A) and transduced cells (B) were taken at 100X magnification 6 days after transduction.

Colorimetric analysis (475 nm absorbance) of LV-hTyrosinase-PGK-Puro transduced cells: A549 and MCF7 cells were transduced with LV-hTyrosinase-PGK-Puro (MOI = 10). After 1 week, cell lysates were collected from parental and transduced cells as well as parental B16F10 cells which endogenously express tyrosinase. To measure tyrosinase expression, 20 μg of lysates were incubated for 4 h with L-DOPA (1.6 mg/mL), which in the presence of tyrosinase is converted to a colored compound with absorbance at 475 nm.