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.
• 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.
• Biosafety level: 1
• Additional notes: FAQs: What resistance does this line carry? This cell line is resistant to Zeocin (from FRT) and Blasticidin (from TR). It is also resistant to hygromycin and puromycin as this has been inherited from the parental RPE1 cell line. When the inventors made this line, they swapped the hygromycin resistance cassette in the original pcDNA5/FRT/TO plasmid for a neomycin resistance marker and so use geneticin (a neomycin analogue) for selection after Flp-In integration. How do you transfect & select cells? The inventors use electroporation and have Invitrogen's Neon transfection system. They leave cells for 2-3 days to recover from transfection, then split them into media containing Geneticin (a neomycin analogue) for selection. Geneticin (910131027, ThermoFisher Scientific) is used in media at a final concentration of 0.5mg/mL. They replace this media every 2-3 days for about 10-14 days, until there is no more cell death. Note that in their standard media we use high quality FBS which should be tet-free. What ratio of plasmids do you use when transfecting? The inventors use 1:4 ratio of pOGG (Flp-recombinase expression) plasmid to the gene of interest plasmid (pcDNA5/FRT/TO/Neo + gene of interest) Do you co-select with additional antibiotics (Blasticidin, zeocin) to keep the Flp-In cassettes? No this isn’t necessary as the FRT and TR are stably integrated.

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: F12:DMEM (1:1) media supplemented with 10% FCS.
• Unit size: 1x10^6 cells / vial
• Shipping conditions: Dry ice
• Storage conditions: Liquid Nitrogen
• Mycoplasma free: Yes

References

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