Species: Human

Cell type: Melanoma

Transgenes: Near-infrared fluorescent protein (iRFP; excitation/emission: 690/713nm) with neomycin resistance (Neo) for selection with G418 and Human sodium iodide symporter (hNIS) with puromycin resistance for selection with puromycin

Media: DMEM, 10% FBS, 1% Pen/Strep, 0.6 mg/mL G418, 1 μg/mL puromycin

Description: A375-iRFP-Neo/hNIS-Puro is a polyclonal population of the human malignant melanoma cell line A375 (ATCC® CRL-1619™) transduced with 1) LV-iRFP-P2A-Neo (LV033) encoding the near-infrared fluorescent protein (iRFP) cDNA under the spleen focus-forming virus (SFFV) promoter linked to the neomycin resistance gene (Neo) via a P2A cleavage peptide and 2) LV-hNIS-P2A-Puro (LV019) encoding the human sodium iodide symporter (hNIS) cDNA under the spleen focus-forming virus (SFFV) promoter linked to the puromycin resistance gene (Puro) via a P2A cleavage peptide.

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-iRFP-Neo/hNIS-Puro cell line has been tested for mycoplasma contamination and is certified mycoplasma free.

Cell Line Authentication: The parental A375 cell line was authenticated and 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 hNIS/iRFP expressing cell line suitable for use as a positive control cell line in iodine uptake and fluorescence assays to verify NIS or iRFP expression respectively in your lentiviral transduced cells.

In vivo: A375 cells form tumors post implantation into immunosuppressed mice. The in vivo growth of these tumors can be monitored using noninvasive, high-resolution 3D PET/SPECT imaging or noninvasive optical imaging.

References on NIS imaging:

1. Fruthwirth et al. A whole body dual modality radionuclide optical strategy for preclinical imaging of metastasis and heterogeneous treatment responses in different. microenvironments. J. Nucl. Med 2014. 55(4): 686-94.

2. Penheiter et al. The sodium iodide symporter (NIS) as an imaging reporter for gene, viral and cell-based therapies. Curr Gene Ther. 2012, 12(1):33-47.

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

NIS Functional Assay (Iodine Uptake): Cells were incubated with ¹²⁵I for 1h in the presence or absence of KClO₄, an inhibitor of NIS-mediated iodine uptake. Radioiodine concentrated within the cells was measured with a gamma counter.

Flow Cytometry for iRFP: A375-iRFP-Neo/hNIS-Puro (red) or isotype control (A375-Fluc-Puro; grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).