Species: Mouse

Cell type: Mammary carcinoma

Transgenes: Firefly luciferase (Fluc) with neomycin resistance (Neo) for selection with G418 and enhanced green fluorescent protein (eGFP) with puromycin resistance (Puro) for selection with puromycin

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

Description: 4T1-Fluc-Neo/eGFP-Puro is a polyclonal population of the murine mammary carcinoma cell line 4T1 (ATCC® CRL-2539™) transduced with  1) LV-Fluc-Neo (LV011) encoding firefly luciferase (Fluc) cDNA under the spleen focus-forming virus (SFFV) promoter linked to the neomycin resistance gene via P2A sequence, and 2) LV-eGFP-PGK-Puro (LV031) encoding enhanced green fluorescent protein (eGFP) cDNA under the spleen focus-forming virus (SFFV) promoter and the puromycin resistance gene under control of the PGK promoter.

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).

Mycoplasma Testing: The 4T1-Fluc-Neo/eGFP-Puro cell line has been tested for mycoplasma contamination and is certified mycoplasma free.

Cell Line Authentication: The parental 4T1 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 luciferase/GFP expressing cell line suitable for use as a positive control cell line in bioluminescence and fluorescence assays to verify luciferase or GFP expression respectively in your lentiviral transduced cells.

In vivo: 4T1 cells form tumors post implantation into syngenic Balb/c mice. The in vivo growth of these tumors can be monitored using noninvasive bioluminescent imaging or optical imaging for GFP fluorescence.

Publications using associated reagents:

LV-eGFP-PGK-Puro (LV031): Shen et al. Immunovirotherapy with vesicular stomatitis virus and PD-L1 blockade enhances therapeutic outcome in murine acute myeloid leukemia. Blood. 2016. March 17: 127(11): 1449-58.

Cell 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.

 

Flow Cytometry for eGFP: 4T1-Fluc-Neo/eGFP-Puro (green) or control (4T1-mNIS-Puro; grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).

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.