R1/E (ATCC® SCRC-1036)

Organism: Mus musculus, mouse  /  Cell Type: embryonic stem cell  /  Tissue: inner cell mass  / 

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Organism Mus musculus, mouse
Tissue inner cell mass
Cell Type embryonic stem cell
Product Format frozen
Morphology Spherical colony
Culture Properties adherent
Biosafety Level 1
Age embryo
Gender male
Strain 129X1 x 129S1
Storage Conditions liquid niitrogen vapor phase
Derivation
The R1/E cell line was subcloned from R1 in EMBL, Heidelberg, Germany by Kristina Vintersten. The R1 cell line was established in August 1991, from a 129X1 x 129S1 3.5 day blastocyst.  
Clinical Data
male
Comments

The R1/E cell line was subcloned from R1 in EMBL, Heidelberg, Germany by Kristina Vintersten. The R1 cell line was established in August 1991, from a 129X1 x 129S1 3.5 day blastocyst. The cells are heterozygous for the c locus (+/c (ch)) and for the pink eye locus (+/p). 

This mouse ES cell line has been shown to be germline competent.In the F1 generation the coat color is uniform agouti, while in the F2 these two coat color genes segregate. The segregation could result in several coat types, from albino, through light brown, to black, depending on the genetic background of the partner of the germline chimaera.

Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development [PubMed: 8378314]. They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras [PubMed: 8361547]. At early passages (up to passage #14), one third of the completely R1-derived newborns generated by tetraploid embryo <-> R1 aggregates survived. No live offspring were produced from cells older than passage #14. 

However, about 20% of subclones derived from passage #14 had the original developmental potential of R1 when tested by tetraploid aggregates [PubMed: 8378314]. R1-derived animals reached adulthood and were fertile. The genetically altered lines derived from R1 gave high efficiency of germline transmission either by injecting them into C57 blastocyst or aggregating them with CD-1 or ICR outbred 8-cell stage embryos. More than 90% of the individual K.O. clones went to germline (n>60) by aggregation chimeras.

Complete Growth Medium Grow ES cells in Mouse ES Cell Basal Medium (ATCC SCRR-2011) that has been supplemented with the following components:
1. 0.1 mM 2-mercaptoethanol (Life Technologies Cat. No. 21985-023)
2. 1,000 U/mL mouse leukemia inhibitory factor (LIF) (Millipore Cat. No. ESG1107)
3. 10% to 15% ES-Cell Qualified FBS (ATCC® SCRR-30-2020) or an ES cell qualified serum replacement
Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C.
Subculturing Subculturing Procedure

Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.

Feeder Cell Preparation for Subcultures

  1. Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
  2. One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.

Dissociation and Transfer of ES Cells

  1. Aspirate the medium from the flask(s) containing ES cells.
  2. Wash with PBS Ca+2/Mg+2-free (ATCC® SCRR-2201).
  3. Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC® 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
  4. Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.
  5. Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
  6. Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm2.
  7. Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.
  8. Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.
Cryopreservation
Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO (ATCC 4-X).
Storage temperature: liquid niitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Name of Depositor A Nagy
Passage History
Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development [PubMed: 8378314]. They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras [PubMed: 8361547]. At early passages (up to passage #14), one third of the completely R1-derived newborns generated by tetraploid embryo <-> R1 aggregates survived. No live offspring were produced from cells older than passage #14. However, about 20% of subclones derived from passage #14 had the original developmental potential of R1 when tested by tetraploid aggregates [PubMed: 8378314]. R1-derived animals reached adulthood and were fertile. The genetically altered lines derived from R1 gave high efficiency of germline transmission either by injecting them into C57 blastocyst or aggregating them with CD-1 or ICR outbred 8-cell stage embryos. More than 90% of the individual K.O. clones went to germline (n>60) by aggregation chimeras.
Year of Origin 1991
References

Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999

Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314

Wood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547

Nagy A, Rossant JProduction and analysis of ES-cell aggregation chimerasIn: Nagy A, Rossant JGene Targeting: A Practical ApproachOxfordOxford University Press177-206, 1999

Basic Documentation
Restrictions

Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact ATCC's Office of Licensing and Business Development at licensing@atcc.org or 703 365 2773.

References

Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999

Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314

Wood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547

Nagy A, Rossant JProduction and analysis of ES-cell aggregation chimerasIn: Nagy A, Rossant JGene Targeting: A Practical ApproachOxfordOxford University Press177-206, 1999