Species: Mouse

Cell type: Lewis Lung Carcinoma

Transgenes: Murine sodium iodide symporter (mNIS) with neomycin resistance (Neo) for selection with G418 and firefly luciferase (Fluc) 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/Fluc-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-Fluc-P2A-Puro (LV012) encoding firefly luciferase (Fluc) cDNA under the spleen focus-forming virus (SFFV) promoter linked to neomycin resistance (Neo) via a P2A cleavage peptide.

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/Fluc expressing cell line suitable for use as a positive control cell line in iodine uptake and bioluminescence assays to verify NIS and luciferase expression respectively in your lentiviral transduced cells.

In vivo: LL/2 cells form tumors post implantation into immunocompromised or syngeneic C57BL/6 mice. The in vivo growth of these metastases can be monitored using non-invasive, high-resolution 3D PET/SPECT imaging with radioiodine or noninvasive bioluminescent imaging with D-luciferin substrate.

NOTE: Host immune selection against the immunogenic firefly luciferase protein may occur in an immunocompetent host, resulting in selection and growth of clones with lower or poor luciferase expression. If that is a concern, please consider using a cell line that expresses mNIS, e.g. LL/2-mNIS-Puro (Imanis catalog #CL054).

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.

 

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.

Bioluminescent images of a subcutaneous LL/2-Fluc-Puro tumor in an athymic mouse

1e7  LL/2-Fluc-Puro cells (Imanis Life catalog #CL050) 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#6867) is shown.