HeLa-Mitotrap-Ap1g1-FKBP
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HeLa-Mitotrap-Ap1g1-FKBP
Cell Line Name
Cell Line 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-Ap1g1-FKBP is a stably transfected HeLa cell line expressing two proteins containing rapamycin binding activity. To generate this cell line, the HeLa-Mitotrap cell line, ECACC 15042201, expressing a single construct expressing a mitochondrial trapping protein containing an FRB domain, (the term FRB refers to protein containing a FKBP12 and Rapamycin Binding (FRB) domains within the mTOR (mammalian Target of Rapamycin) protein, has been transfected with a second construct expressing siRNA-resistant subunits, FKBP-tagged subunits of the adaptor protein (AP1) complexes of clatharin-coated vesicles (CCVs) together with an FKBP and rapamycin-binding domain with a mitochondrial targeting signal. An FKBP domain, FKBP is a cellular chaperone protein and prolyl isomerase which is a target for rapamycin binding and will dimerize with FRB-containing protein. Upon treatment with rapamycin a target protein containing an FKBP domain, e.g, AP1, will dimerize with FRB domains and sequester the resulting protein complexes 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.The α-FKBP construct was based on a pIRESneo2 plasmid encoding an siRNA-resistant form of a, which includes a brain-specific insert that acts as an epitope tag (Motley et al., 2006, PMID: 17035630). The FKBP coding sequence was amplified by PCR and inserted into the linker region of α. The open reading frame was sequenced, the plasmid was transfected into HeLaM cells, and clonal cell lines were isolated as previously described (Motley et al., 2006).
General Info
Species
Human
Links
Cellosaurus: CVCL_AR67
Release Conditions
Characteristics
Tissue of Origin
Morphology
Applications
Study of protein function by rapid protein inactivation
Disease
Culture Conditions
Cell Type
Subculture Routine
Trypsinise cultures at 80% confluence with 0.05% Trypsin/EDTA. Seed flasks at 2x10⁴ cells/ cm². Cultures must be incubated in a humidified 10% CO₂/90% air incubator at 37°C.
Culture Medium
Dulbecco's Modified Eagle's Medium, high glucose (DMEM) + 2mM Glutamine + 10% Foetal Bovine Serum (FBS)
Growth Mode
Additional Info
Depositor
Country of Origin
GMO Status
Hazard Group (ACDP)
Applications
References
Robinson MS, Sahlender DA, Foster SD. Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Dev Cell. 2010 Feb 16;18(2):324-31. doi: 10.1016/j.devcel.2009.12.015. PMCID: PMC2845799, PMID: 20159602Hirst J, Borner GH, Antrobus R, Peden AA, Hodson NA, Sahlender DA, Robinson MS. Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system. Curr Biol. 2012 Sep 25;22(18):1711-6. doi: 10.1016/j.cub.2012.07.012. Epub 2012 Aug 16. PMCID: PMC3485558
Documents
Images
Available Formats
- Frozen
- DNA-5µg (100ng/µl)
Citation Guidance
If use of this culture results in a scientific publication, it should be cited in the publication as: HeLa-Mitotrap-Ap1g1-FKBP (ECACC 15042203).
Biosafety Information
Unless specified otherwise, at ECACC we routinely handle all of our cell lines at containment level 2 in accordance with the ACDP guidelines (Advisory Committee on Dangerous Pathogens) (UK). All cell cultures have the potential to carry as yet unidentified adventitious agents. It is the responsibility of the end user to ensure that their facilities comply with biosafety regulations for their own country. ACDP Guidance: Biological agents: Managing the risks in laboratories and healthcare premises.
Further Information
The Culture Collections represent deposits of cultures from world-wide sources. While every effort is made to ensure details distributed by Culture Collections are accurate, Culture Collections cannot be held responsible for any inaccuracies in the data supplied. References where quoted are mainly attributed to the establishment of the cell culture and not for any specific property of the cell line, therefore further references should be obtained regarding cell culture characteristics. Passage numbers where given act only as a guide and Culture Collections does not guarantee the passage number stated will be the passage number received by the customer.
Cultures supplied by Culture Collections are for research purposes only. Enquiries regarding the commercial use of a cell line are referred to the depositor of the cell line. Some cell lines have additional special release conditions such as the requirement for a material transfer agreement to be completed by the potential recipient prior to the supply of the cell line. Please view the Terms & Conditions of Supply for more information.