Species: Mouse

Cell type: Lewis Lung Carcinoma

Transgenes: Murine sodium iodide symporter (mNIS) with neomycin resistance (Neo) for selection with G418 and enhanced green fluorescent protein (eGFP) with puromycin resistance (Puro) for selection with puromycin

Media: DMEM, 10% FBS, 1% Pen/Strep, 1.25mg/mL G418, 2μg/mL puromycin

Description: LL/2-mNIS-Neo/eGFP-Puro is a polyclonal population of the Lewis lung carcinoma cell line LL/2 (ATCC® CRL-1642™), also commonly known as LLC1, transduced with LV-mNIS-P2A-Neo (LV025) encoding the murine sodium iodide symporter (mNIS) cDNA under the spleen focus-forming virus (SFFV) promoter linked to the neomycin resistance gene (Neo) via a P2A cleavage peptide and LV-eGFP-PGK-Puro (LV031) encoding enhanced green fluorescent protein (eGFP) cDNA under the spleen focus-forming virus (SFFV) promoter and the puromycin resistance gene (Puro) under control of the PGK promoter.

The lentiviral vectors used are self-inactivating (SIN) vectors 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 LL/2 cell line was authenticated and certified free of interspecies cross-contamination by short tandem repeat (STR) profiling with 27 STR loci.

Recommended uses:

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

In vivo: LL/2 cells form tumors post implantation into immunocompromised or syngenic C57BL/6 mice. The in vivo growth of these metastases can be monitored using non-invasive, high-resolution 3D PET/SPECT imaging with radioiodine. In vivo imaging for GFP fluorescence is not recommended due to high background autofluorescence. Tissues may be harvested post mortem for analysis by conventional microscopy.

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)

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

Flow Cytometry for eGFP: LL/2-mNIS-Neo/eGFP-Puro (green) or control (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.