High-titer purified LV-DsRed-PGK-Puro stocks

Promoters: 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.

Transgenes: Red fluorescent protein (DsRed) and the puromycin resistance gene (Puro)

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

Construct: This is a ready-to-use lentivirus preparation. The virus encodes the red fluorescent protein (DsRed) 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 2e7 TU/mL (by qPCR titration of transduced HeLa H1 cells)

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

Vector Map:

Fluorescence Expression: HeLaH1 cells were transduced with LV-DsRed-PGK-Puro (MOI = 10) and after three days the cells were amplified under puromycin selection. The transduced cells (red) and untransduced control cells (grey) were fixed with paraformaldehyde and analyzed by flow cytometry.