Hepa1-6-Fluc-Neo is a polyclonal population derived of the hepatoma Hepa1-6 cell line (ATCC® CRL-1830™). To achieve stable reporter expression in the polyclonal population, parental Hepa1-6 cells were transduced with LV-Fluc-P2A-Neo (LV011) and selected using G418. LV-Fluc-P2A-Neo encodes the firefly luciferase (Fluc) cDNA linked to the neomycin resistance gene (Neo) via a P2A cleavage peptide under the spleen focus-forming virus (SFFV) promoter.

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

The parental Hepa1-6 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)

Luciferase Expression

 10⁴, 10⁵, or 10⁶ cells were placed in wells of a 96-well plate and 0.3 mg of d-luciferin was added. The plate was immediately imaged using a Xenogen IVIS Spectrum.

Complete Growth Medium: DMEM supplemented with 10% FBS, 1X Penicillin/Streptomycin, and 1.25 mg/mL G418.

The addition of G418 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.

These cells are suitable for in vitro and in vivo experimentation. Hepa1-6 cells form primary tumors and spontaneous lung metastases post implantation into syngenic C57L/J mice^1,2. However, tumor formation in syngenic mice is highly variable, so for the most consistent results immunocompromised mice are recommended for studies.

The Fluc transgene facilitates in vivo noninvasive bioluminescent imaging of implanted cells. 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:
¹Lei and Ling. World J Gastroenterol 2015. 21: 10137-10149.
²Wang et al. Cell Physiol Biochem 2016. 38: 306-318.
³Miyoshi et al. J Virol 1998. 72:8150-8157.