Species: Mouse

Cell type: Mammary carcinoma

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

Media: RPMI, 10% FBS, 1% Pen/Strep, 0.1 mg/mL G418, 2 μg/mL puromycin

Description: 4T1-mNIS-Puro/Fluc-Neo is a polyclonal population of the murine mammary carcinoma cell line 4T1 (ATCC® CRL-2539™) transduced with 1) LV-mNIS-PGK-Puro (LV022) encoding murine sodium iodide symporter (mNIS) cDNA under the spleen focus-forming virus (SFFV) promoter and the puromycin resistance gene (Puro) under control of the PGK promoter, and 2) 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 sequence. 

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 4T1 cell line was authenticated and cells 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 mNIS/Fluc expressing cell line suitable for use as a positive control cell line in iodine uptake and bioluminescence assays to verify NIS or luciferase expression respectively in your lentiviral transduced cells.

In vivo: 4T1 cells form tumors post implantation in mice. The in vivo growth of these tumors can be monitored using noninvasive, high-resolution 3D PET/SPECT imaging 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)

Transgene Validation:

NIS function: NIS mediated iodine uptake assay in the presence or absence of KClO4, a competitive inhibitor of uptake.

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 4T1-Fluc-Neo tumor in an athymic mouse. 

10⁷ 4T1-Fluc-Neo cells (Imanis Life catalog #CL020) 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 16 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#3876) is shown.