Cancer Research Technology
Log in Register
Menu

HeLa-Mitotrap-GGA2-FKBP Cell Line

Invented by Margaret Robinson , Jennifer Hirst
Invented at University of Cambridge

Info

Antigen/Gene or Protein Targets GGA2 (Golgi-associated gamma adaptin)
Parental Line HeLa
Host Human
Tissue Cervix
Disease Keywords Protein transport; cell trafflicking machinery; hereditary spastic paraplegias
Relevance The HeLa-Mitotrap-GGA2-FKBP cell line has been designed to provide new insights into the the trafficking of proteins between the trans-Golgi network and the lysosome.
This type of approach, a “knocksideways,” inactivates proteins by using a small molecule to trap them onto mitochondria. It is typically 3 to 4 orders of magnitude faster than a knockdown.
The HeLa-Mitotrap-GGA2-FKBP cell line was generated by transfecting the HeLa-Mitotrap cell line with a second construct expressing FKBP-tagged subunits of GGA2. An FKBP domain is a cellular chaperone protein and prolyl isomerase which is a target for rapamycin binding and dimerizes with FRB-containing protein. Upon treatment with rapamycin the target protein containing an FKBP domain, (e.g, GGA2), will dimerize with FRB domains and the resulting protein complexes will be sequestered to the mitochondria within minutes of treatment rendering the cells ready for immediate assay.
Production Details HeLa-Mitotrap expresses a mitochondrial trapping construct with an FRB domain and is maintained in hygromycin-containing medium. The other three cell lines also express Mitotrap, and in addition they express a protein of interest with an FKBP domain attached, which can be trapped onto mitochondrial when rapamycin is added. The three proteins of interest are Ap1g1 or gamma-adaptin; Ap2a2 or alpha-adaptin isoform C; and GGA2.
Conditional Yes
Conditional Description Rapamicin-induced knocksideways
Research Area Cell Signaling & Signal Transduction, Cellular Transport
Recommended Growing Conditions HeLa cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C and 5% CO2. Cells were transfected using GeneJuice. Rapamycin was used at 200 nM, vehicle was ethanol (0.1%). MLN8237was used at 1 µM, vehicle was DMSO (0.01%).
Cellosaurus ID CVCL_AR70

References

There are 4 reference entries for this reagent.

View All References

References: 4 entries

Cheeseman et al. 2013. J Cell Sci. 126(Pt 9):2102-13. PMID: 23532825.

Hirst et al. 2012. Curr Biol. 22(18):1711-6. PMID: 22902756.

Mir et al. 2014. Methods Mol Biol. 1121:3-23. PMID: 24510808.

Robinson et al. 2010. Dev Cell. 18(2):324-31. PMID: 20159602.


Add a reference

References: 4 entries

Cheeseman et al. 2013. J Cell Sci. 126(Pt 9):2102-13. PMID: 23532825.

Hirst et al. 2012. Curr Biol. 22(18):1711-6. PMID: 22902756.

Mir et al. 2014. Methods Mol Biol. 1121:3-23. PMID: 24510808.

Robinson et al. 2010. Dev Cell. 18(2):324-31. PMID: 20159602.


Add a reference

Inventor Information

Inventors

Margaret Robinson

Margaret Robinson

Jennifer Hirst

Jennifer Hirst

Add an inventor