CT26.WT-iRFP-Neo/mNIS-Puro is a polyclonal population of the mouse colorectal carcinoma cell line CT26.WT (ATCC® CRL-2638™). To achieve stable reporter expression in the polyclonal population, parental CT26.WT cells were transduced with LV-iRFP-P2A-Neo (LV033) and LV-mNIS-PGK-Puro (LV022) and selected using G418 and puromycin. LV-iRFP-P2A-Neo encodes the near-infrared fluorescent protein (iRFP) cDNA linked to the neomycin resistance gene (Neo) via a P2A cleavage peptide (P2A) under the spleen focus-forming virus (SFFV) promoter. LV-mNIS-PGK-Puro (LV022) encodes mouse sodium iodide symporter (mNIS) cDNA under the SFFV promoter and the puromycin resistance gene (Puro) under the phosphoglycerate kinase (PGK) promoter.

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

The parental CT26.WT cell line has been authenticated and certified free of interspecies cross contamination by short tandem repeat (STR) profiling with 9 STR loci.

Due to the immunogenicity of the reporter genes in this cell line, we recommend using immunocompromised mice for in vivo studies.

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 NIS mediated iodine uptake. Radioiodine concentrated within the cells was measured with a gamma counter.

iRFP Expression

 CT26.WT-iRFP-Neo/mNIS-Puro cells (red) or control (CT26.WT-Fluc-Neo; 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, 3 µg/mL puromycin, and 0.4 mg/mL G418.

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.

CT26.WT cells easily detach from tissue culture surfaces. Coating culture plates with poly-D-Lysine prior to use is recommended for applications involving frequent washes, culture media changes, or rocking.

These cells are suitable for in vitro and in vivo experimentation. CT26.WT cells form tumors and pulmonary metastases post implantation into syngenic Balb/c mice.¹

The iRFP and NIS transgenes facilitate noninvasive bioluminescence and 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 LTR². 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:
¹Wang et al. J Immunol. 1995. 154:4685-4692.
²Miyoshi et al. J Virol 1998. 72:8150-8157.