RPE1 FRT/TR Cell Line

Tool Details

*FOR RESEARCH USE ONLY (for other uses, please contact the licensing team)

• Name: RPE1 FRT/TR Cell Line
• Research fields: Genetics
• Parental cell: RPE1
• Organism: Human
• Tissue: Eye
• Growth properties: Adherent cell line
• Conditional: Yes
• Conditional description: Tetracycline-inducible expression of genes of interest.
• Description: This cell line can be used to generate cell lines with tetracycline inducible expression of genes of interest. The cell line has a randomly integrated FRT locus and Tet repressor for Flp-In expression. For example the originator of the cell line used this cell line to generate tetracycline-inducible cell lines expressing APC15-IRES2-mRuby, APC15-3xflag and Cyclin B1-L45A-HA using the FLIP-in system and a modified pCDNA5/FRT/TO vector. This cell line is resistant to Zeocin (from FRT) and Blasticidin (from TR). It also carries resistance to hygromycin and puromycin, inherited from the parental RPE1 cell line. After Flp-In integration, selection is performed using Geneticin (a neomycin analogue), because the hygromycin resistance cassette in the original pcDNA5/FRT/TO plasmid was replaced with a neomycin resistance marker. The FRT and TR cassettes are stably integrated, so additional co-selection with Blasticidin or Zeocin is not required.
• Production details: This tetracycline-inducible RPE1 cell line was created by random integration of an FRT site and a Tet repressor gene into retinal pigment epithelial 1 (RPE1) cells. The inventors used electroporation for transfection, specifically with Invitrogen’s Neon transfection system. Cells were allowed to recover for 2 –3 days post-transfection, then split into media containing Geneticin at a final concentration of 0.5 mg/mL (ThermoFisher Scientific, 910131027). This selection medium was refreshed every 2–3 days for approximately 10–14 days until cell death ceases. Standard media included high-quality, tetracycline-free FBS. For plasmid ratios during transfection, a 4:1 ratio of pOG44 (Flp-recombinase expression plasmid) to the gene-of-interest plasmid (pcDNA5/FRT/TO/Neo + gene of interest) was used.
• Biosafety level: 1
• Cellosaurus id: CVCL_VP32

Applications

• Application notes: This cell line is resistant to Zeocin (FRT), Blasticidin (TR) and Puromycin (hTERT). The hygromycin resistance cassette in the original plasmid was replaced with neomycin resistance. For selection after the Flp-In integration the inventors used Geneticin G418 sulphate (an analog of neomycin sulphate) at a final concentration of 0.5 mg/mL.

Handling

• Format: Frozen
• Growth medium: 500 mL Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (Sigma: D6241), 17.3 mL of 7.5% sodium bicarbonate, 10% FBS, 1% Pen/Strep, 1% 200 mM Glutamax and 1 mL Amphotericin B.
• Unit size: 1x10^6 cells / vial
• Shipping conditions: Dry ice
• Storage conditions: Liquid Nitrogen
• Subculture routine: Please see detailed protocol within the Product Datasheet in the Documentation section below.
• Mycoplasma free: Yes

References

• Mansfield et al. 2011. Nat Cell Biol. 13:1234-43. PMID: 21926987

Documentation

• Product Datasheet