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ES-R1-EGFP B5/EGFP

ES-R1-EGFP B5/EGFP

Catalogue No.

07072007

Cell Line Name

ES-R1-EGFP B5/EGFP

Cell Line Description

Pluripotent mouse embryonic stem cell line expressing GFP. This green fluorescent variant was generated by the random integration of EGFP transgenes into ES-R1 (ECACC cat no. 07072001) using co-electroporation with a circluar selectable marker containing vector pPGK Puro. The vector is driven by a CMV immediate early enhancer coupled to the chicken beta-actin promoter and first intron.

General Info

Species

Mouse

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

Embryo

Morphology

Spheroidal

Applications

Embryonic Stem (ES) cell-based genetic manipulations and lineage development of ES cells. Easily visualised Green Fluorescent Protein (GFP) marker for chimeric studies and sources of marked cells or tissues for transplantation/explant studies

Disease

None Stated

Culture Conditions

Cell Type

Adherent monolayer of spheroidal cells on feeder layer of mouse primary embryonic fibroblasts

Subculture Routine

Embryonic stem (ES) cells require the use of mitotically inactivated feeder cells to support the growth of stem cells in the undifferentiated state. Mouse embryonic fibroblasts, STO (ECACC 86032003) or SNL 76/7 (ECACC 07032801) can be used. At ECACC plastic ware is pre-coated with gelatine prior to plating feeder cells.Porcine gelatine (Sigma G1890) is dissolved in sterile water (0.5g/500ml) at 56°C. The 0.1% solution is sterilized by filtration (0.22µm). Add 0.1% gelatine to plastic ware to cover bottom, and incubate for 20 minutes at room temperature.  Remove gelatine, wash with PBS once and replace with appropriate culture medium. The flask/dish must not be allowed to dry out.Feeder layers are prepared on the gelatinized flasks at least 24 hours in advance of being required. An ampoule is thawed in 37°C water bath and the contents quickly transferred to a 15ml centrifuge tube. MEF medium is added drop wise to 5ml. Cells are centrifuged at 150 x g for 5 minutes at Room Temperature (RT). Cells are resuspended in 5ml of MEF medium. Cells are counted and added to flasks containing the correct medium at 1-3 x 10⁴ cells/cm².An ampoule of ES cells is thawed in 37°C water bath and the contents quickly transferred to a 15ml centrifuge tube. KSR medium is added drop wise to 5ml. Cells are centrifuged at 150 x g for 5 minutes. Cells are resuspended in 5ml of KSR medium. The prepared feeder flask is washed once with PBS and KSR medium added. ES cells should be plated at 4-5 x 10⁴ cells/cm².  Cultures must be incubated in a humidified 5% CO₂/95% air incubator at 37°C. A 100% media change must be performed every day and cells passaged every 2-3 days. Colonies must not be allowed to touch each other as overgrowth will result in differentiation.

Culture Medium

MEF medium consists of Advanced DMEM/F12 (Invitrogen 12634010), 10% FBS (Perbio SH30070.03E), 2 mM Glutamine (Invitrogen 25030024) and 0.1 mM β-mercaptoethanol (Sigma M6250).KSR medium consists of KO-DMEM (Gibco 10829), 20% Knock-Out Serum Replacer (Gibco 10828), 2 mM Glutamine (Invitrogen 25030024), NEAA (Invitrogen 11140035), 0.1 mM β-mercaptoethanol (Sigma M6250) and LIF 1000 Units/ml (ESGRO ESG1106).

Growth Mode

Adherent

Additional Info

Depositor

Dr A Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, M5G 1X5, Canada.

Country of Origin

Canada

GMO Status

Genetically Modified Organism Class 1 (GMO1)

Hazard Group (ACDP)

Hazard Group (ACDP) 2

Applications

References

Hadjantonakis & Nagy (2000) FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis 27: 95-98. PMID: 10951501.

Bibliography

Hadjantonakis & Nagy (2001) The color of mice: in the light of GFP-variant reporters. Histochem Cell Biol 115: 49-58. PMID: 11219608. Hadjantonakis et al., (2002) Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal. BMC Biotechnology 2:11. PMID: 12079497. Hadjantonakis et al., (2003) Technicolour transgenics: imaging tools for functional genomics in the mouse. Nature Rev Genet 4: 613-625. PMID: 12897773.

Available Formats

  • Frozen
  • DNA-5µg (100ng/µl)

If use of this culture results in a scientific publication, it should be cited in the publication as: ES-R1-EGFP B5/EGFP (ECACC 07072007).

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.