Species: Human

Cell type: Melanoma

Transgenes: Enhanced green fluorescent protein (eGFP) with puromycin resistance (Puro) for selection with puromycin

Media: DMEM, 10% FBS, 1% Pen/Strep, 1 μg/mL puromycin

Description: A375-eGFP-Puro is a polyclonal population of the human malignant melanoma cell line A375 (ATCC® CRL-1619™) transduced with LV-eGFP-PGK-Puro (LV031) encoding the enhanced green fluorescent protein (eGFP) cDNA under the spleen focus-forming virus (SFFV) promoter and the puromycin resistance gene (Puro) under the mouse phosphoglycerate kinase (PGK) promoter.

The lentiviral vector used is a self-inactivating (SIN) vector in which the viral enhancer and promoter has 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).

Mycoplasma Testing: The A375-eGFP-Puro cell line has been tested for mycoplasma contamination and is certified mycoplasma free.

Cell Line Authentication: The parental A375 cell line was authenticated are certified free of interspecies cross-contamination by short tandem repeat (STR) profiling with 9 STR loci including CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, vWA and sex chromosome marker Amelogenin.

Recommended uses: In vitro: This is a high GFP expressing cell line suitable for use as a positive control cell line in fluorescence assays to verify GFP expression in your lentiviral transduced cells. In vivo: A375 cells form tumors post implantation into immunosuppressed mice. 

Note: In-life imaging for GFP fluorescence is not recommended due to high background autofluorescence and low light penetration. Tissues may be harvested post mortem for analysis by conventional fluorescence microscopy. For in vivo imaging, please use luciferase or the nonimmunogenic NIS reporter gene.

Morphology: Low- and high-density cell morphology (200X)

 Flow Cytometry for eGFP: A375-eGFP-Puro (green) or control (A375-Fluc-Puro; grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).

 

 

 

 

 

 

 

 

 

Publications that used associated reagents: 

LV-eGFP-PGK-Puro (LV031): Shen et al. Immunovirotherapy with vesicular stomatitis virus and PD-L1 blockade enhances therapeutic outcome in murine acute myeloid leukemia. Blood. 2016. March 17: 127(11): 1449-58.