Species: Mouse

Cell type: Lewis Lung Carcinoma

Transgenes: Firefly luciferase (Fluc) with puromycin resistance (Puro) for selection with puromycin

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

Description: LL/2-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-Fluc-P2A-Puro (LV012) encoding the firefly luciferase (Fluc) 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).

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 Fluc expressing cell line suitable for use as a positive control cell line in bioluminescence assays to verify luciferase expression in your lentiviral transduced cells.

In vivo: LL/2 cells form tumors post implantation into mice. The in vivo growth of primary tumors or metastases can be monitored using bioluminescent imaging with D-luciferin substrate.

NOTE: LL/2-Fluc tumors may be grown in C57BL/6 immunocompetent mice but host immune selection against the immunogenic firefly luciferase protein may occur, resulting in selection and growth of clones with lower or poor luciferase expression.

For in vivo imaging in immunocompetent hosts, please use the nonimmunogenic murine NIS reporter gene.

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.

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

1e7  LL/2-Fluc-Puro cells 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.