Description
This cell line can be used to determine the function of a protein by rapidly inactivating the protein of interest by rapamycin induced rerouting of the protein to the mitochondria. The HeLa-Mitotrap cell line is a stably transfected HeLa cell line expressing a mitochondrial trapping protein containing an FRB domain. The term FRB refers to a protein containing FKBP12 and Rapamycin Binding (FRB) domains within the mTOR (mammalian Target of Rapamycin) protein. The Hela-Mitotrap cell line can be transfected to express the target protein of interest genetically modified to have an FKBP domain. Upon treatment of the cells with rapamycin target proteins containing an FKBP domain will dimerize with FRB domains. The resulting protein complexes are sequestered to the mitochondria. Treatment of cell lines containing both FRB and FKBP rapamycin binding proteins can sequester target proteins to the mitochondria within minutes of treatment rendering the cells ready for immediate assay. This type of approach, referred to as knocksideways, should be widely applicable as a means of inactivating proteins within a timescale of seconds or minutes rather than days. The derivation of the expression construct for the generation of the FRB expressing HeLa cell line is described in Motley et.al (2006, PMID 17035630).The Mito-YFP-FRB construct was based on a pEYFP-FRB plasmid (O. Glebov, MRC Laboratory of Molecular Biology, Cambridge, UK). The N-terminal sorting signal of Tom70p was amplified by PCR from yeast genomic DNA and cloned into the plasmid, then the Mito-YFP-FRB coding sequence was moved into the retroviral vector pQCXIH (Clontech), which carries a hygromycin resistance gene. Stable cell lines were selected using hygromyin selection.
Adherent Suspension
Adherent
Suggested Medium
Dulbecco's Modified Eagle's Medium, high glucose (DMEM) + 2mM Glutamine + 10% Foetal Bovine Serum (FBS)
Quality Control
Cell lines were tested and found to be free of mycoplasma, bacterial, viruses, and other toxins. All cells were above 95% viability before freezing.
Morphology
Epithelial, polygonal
Shippment
The cells are shipped using dry ice.
Storage
Store the cell lines in liquid nitrogen vapor (less than -130°C)
Warranty
Our cell products are for research use only. They are not authorized for human, in vitro diagnostic procedures or for therapeutic procedures.
