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UK Health Security Agency

HEK293A GFP-LC3

HEK293A GFP-LC3

Catalogue No.

14050801

Cell Line Name

HEK293A GFP-LC3

Cell Line Description

A stable cell line expressing EGFP-tagged LC3 (rat) in background of HEK293 cell line. This reporter cell line can be used to monitor induction of autophagy after amino acid starvation or rapamycin treatment as well as autophagosome formation. Also, this cell line provides a useful tool for the study of autophagy using GFP-LC3 as a standard assay read out by both immunofluorescence and biochemical methods.To generate the cell line EGFP-LC3 (rat) plasmid was transfected into HEK293A cells. The cells were selected with 800ug/ml Geneticin. Single cell clones were selected and grown up. Several clones were tested and clone 2GL9 was taken as the best based on a number of parameters (including GFP-LC3 expression levels and responsiveness to amino acid deprivation).Description of the genetic modification to generate this cell line: HEK293 cells transfected with a GFP-LC3 (green fluorescent protein-microtubule-associated protein 1 light chain 3) plasmid. cDNA encoding rat LC3 was obtained by RT–PCR from rat brain total cDNA and subcloned into the EcoRI site of the eukaryotic expression vector pCI-neo (Promega, Madison, WI). It was then excised and the LC3 cDNA inserted into the BglII and EcoRI sites of pEGFP-C1, a GFP fusion protein expression vector (Clontech Laboratories), Kabeya Y, et.al. (2000).

General Info

Species

Human

Release Conditions

Restricted - commercial organisations are required to complete the 'Cell Line Release Authorisation for Research Use in Commercial Organisations' release conditions form in the supporting documents section.

Characteristics

Tissue of Origin

Kidney (embryo)

Morphology

Small slightly rounded cells growing in a dispersed manner

Disease

None Stated

Culture Conditions

Cell Type

Neuronal

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). Geneticin at (400 µg/ml) can be added for maintenance but this is not normally necessary as the GFP-LC3 expression level is maintained over at least 20 passages.

Growth Mode

Adherent

Additional Info

Depositor

Cancer Research Technology Limited. Cancer Research UK funded research by Sharon Tooze, Ph.D. Secretory Pathways Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln'ÂÂs, Inn Field, London WC2A 3PX, UK.

Country of Origin

United Kingdom

GMO Status

Genetically Modified Organism Class 1 (GMO1)

Hazard Group (ACDP)

Hazard Group (ACDP) 2

Applications

References

Kochl R, Hu XW, Chan EY, Tooze SA. Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. Traffic. 2006 Feb;7(2):129-45. PMID: 16420522.

Bibliography

Chan EY, Longatti A, McKnight NC, Tooze SA. Kinase-inactivated ULK proteins inhibit autophagy via their conserved C-terminal domains using an

Available Formats

  • Frozen

If use of this culture results in a scientific publication, it should be cited in the publication as: HEK293A GFP-LC3 (ECACC 14050801).

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.

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.