Species: Mouse

Cell type: Melanoma

Transgenes: Firefly luciferase (Fluc) with neomycin resistance (Neo) for selection with G418 and murine sodium iodide symporter (mNIS) with puromycin resistance (Puro) for selection with puromycin

Media: DMEM, 10% FBS, 1% Pen/Strep, 0.8mg/mL G418, 1 µg/mL puromycin

Description: B16F10-Fluc-Neo/mNIS-Puro is a polyclonal population of the murine melanoma cell line B16F10 (ATCC^® CRL-6475^™) transduced with 1) LV-Fluc-P2A-Neo (LV011) encoding the firefly luciferase (Fluc) cDNA under the spleen focus-forming virus (SFFV) promoter linked to the neomycin resistance gene (Neo) via a P2A cleavage peptide and 2) LV-mNIS-PGK-Puro (LV022) encoding the murine sodium iodide symporter (mNIS) cDNA under the SFFV promoter and the puromycin resistance gene (Puro) under control of the 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).

Cell Line Authentication: The parental B16F10 cell line was authenticated and are certified free of interspecies cross-contamination by short tandem repeat (STR) profiling with 27 STR loci.

Recommended uses:

In vitro: This is a high Fluc/mNIS expressing cell line suitable for use as a positive control cell line in bioluminescence assays and iodine uptake assays to verify luciferase or NIS expression respectively in your lentiviral transduced cells.

In vivo: B16F10 cells form metastases in the lungs of mice. The in vivo growth of these metastases can be monitored using noninvasive, high-resolution 3D PET/SPECT imaging with the NIS reporter gene or bioluminescent imaging with D-luciferin substrate.

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.

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

Luciferase Assay: 10⁴, 10⁵, or 10⁶ cells were placed in wells of a 96-well plate and 0.3 mg of d-luciferin was added to the indicated wells. The plate was immediately imaged using a Xenogen IVIS Spectrum.

NIS Function Assay (Iodine Uptake): Cells were incubated with I-125 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.

Bioluminescent images showing growth of a subcutaneous B16F10-Fluc tumor in an athymic mouse

10⁷ B16-Fluc-Puro cells (Imanis Life catalog#CL052) were injected subcutaneously into the right flanks of female NCR athymic mice. Mice were imaged at day 0 on the day of cell implantation and at day 7 using a Perkin Elmer IVIS® Spectrum system, at 10-15 minutes post intraperitoneal injection of D-luciferin at 150 mg/kg. Tumor size was measured using calipers. Data from a representative mouse (ID#6383) is shown.