Species: Human

Cell type: Colorectal carcinoma

Transgenes: Human sodium iodide symporter (hNIS) with neomycin resistance (Neo) for selection with G418 and firefly luciferase (Fluc) with puromycin resistance for selection with puromycin

Media: DMEM, 10% FBS, 1% Pen/Strep, 0.5 mg/mL G418, 1 μg/mL puromycin

Description: HCT116-hNIS-Neo/Fluc-Puro is a polyclonal population of the human colorectal carcinoma cell line HCT116 (ATCC® CCL-247™) transduced with 1) LV-hNIS-Neo (LV013) encoding the human sodium iodide symporter (hNIS) cDNA under the spleen focus-forming virus (SFFV) promoter linked to the neomycin resistance gene (Neo) via an IRES and 2) LV-Fluc-P2A-Puro (LV012) encoding firefly luciferase (Fluc) cDNA under the 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).

Mycoplasma Testing: This cell line has been tested for mycoplasma contamination and is certified mycoplasma free.

Cell Line Authentication: Authentication of the parental HCT116 cell line was performed by short tandem repeat (STR) profiling with 9 STR loci including CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, vWA and sex chromosome marker Amelogenin. STR profiling of HCT116 cells are verified and there is no interspecies cross contamination detected.

Recommended uses:

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

In vivo: This human colorectal cell line grows as xenografts in immunocompromised mice. Growth of these tumors can be monitored using noninvasive high-resolution 3D SPECT or PET imaging or bioluminescent imaging using D-luciferin substrate.

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 HCT116-Fluc-Puro tumor in an athymic mouse

10⁷ HCT116-Fluc-Puro cells (Imanis Life catalog #CL008) 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 days 7 and 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#4772) is shown.