HCT116-hNIS-Neo/iRFP-Puro is a polyclonal population of the human colorectal carcinoma cell line HCT116 (ATCC® CCL-247™). To achieve stable reporter expression in the polyclonal population, parental HCT116 cells were transduced with LV-hNIS-Neo (LV013) and LV-iRFP-P2A-Puro (LV032) and selected using G418 and puromycin. LV-hNIS-Neo encodes the human sodium iodide symporter (hNIS) cDNA linked to the neomycin resistance gene (Neo) via an internal ribosomal entry site (IRES) under the spleen focus-forming virus (SFFV) promoter. LV-iRFP-P2A-Puro encodes the near-infrared fluorescent protein (iRFP) cDNA linked to the puromycin resistance gene (Puro) via a P2A cleavage peptide under the SFFV promoter.

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

The parental HCT116 cell line has been authenticated and certified free of interspecies cross contamination by short tandem repeat (STR) profiling with 9 STR loci.

Replication Competent Lentivirus (RCL) Test (including a test report) is available for this cell line at an added cost.  Contact us to learn more.

Morphology

Low and high density cell morphology (200x)

NIS Expression

Cells were incubated with I-125 for one hour in the presence or absence of KClO₄, an inhibitor of iodine uptake. Radioiodine concentrated within the cells was measured with a gamma counter.

iRFP Expression

HCT116-hNIS-Neo/iRFP-Puro (red) or control (HCT116; grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).

Complete Growth Medium: DMEM supplemented with 10% FBS, 1X Penicillin/Streptomycin, 0.5 mg/mL G418, and 1 µg/mL puromycin.

The addition of G418 and puromycin to the complete growth medium maintains high dual reporter expression over continued passage of the cells. It is highly recommended, especially if the cells undergo multiple passages prior to being used for studies.

These cells should be grown in the indicated medium and passaged when they reach confluency. For routine passaging, cells are recommended to be split at a 1:10 ratio every 3-4 days.

HCT116 cells can detach from tissue culture surfaces upon repeated washing. Coating culture plates with poly-D-Lysine prior to use is recommended for applications involving frequent washes or culture media changes.

These cells are suitable for in vitro and in vivo experimentation. HCT116 cells are a xenograph model for colorectal carcinoma and will form primary tumors and distant metastases (lung, liver, and lymph nodes) post-implantation into immunosupressed mice.^1,2 The iRFP and NIS transgenes facilitate noninvasive in vivo and ex vivo fluorescence imaging and high-resolution 3D SPECT/PET imaging, respectively, of implanted cells.To reduce background autofluorescence, mice should be fed an alfalfa-free diet for at least a week prior to imaging.

The cells can be amplified in vitro and used to generate additional frozen stocks. Cryopreservation of low passage stocks is recommended. Frozen stocks should be preserved in a designated cryopreservation medium.

These cells were generated via lentiviral vector transduction. The lentiviral vector used for transduction was a self-inactivating (SIN) vector in which the viral enhancer and promoter have 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 LTR³. Nevertheless, all work with these cells should be performed under biosafety-level 2 (BSL2) conditions by trained personnel. Institutional requirements may permit handling of these cells under BSL1 conditions if certain criteria are met.

References:
¹Brattain et al. Cancer Res. 1981. 41:1751-1756.
²Yang et al. Anticancer Res. 1997. 17:3463-3468.
³Miyoshi et al. J Virol 1998. 72:8150-8157.