R1 (ATCC® SCRC-1011)

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


[Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office (2007). The entire text is available online at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.]

 

Age embryo, blastocyst
Gender Male
Strain 129X1 x 129S1
Storage Conditions liquid nitrogen vapor phase
Karyotype The cells are heterozygous for the c locus (+/c (ch)) and for the pink eye locus (+/p). 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.
Derivation

The R1 cell line was established in August 1991, from a 3.5 day blastocyst produced by crossing two 129 substrains (129S1/SvImJ and 129X1/SvJ).


Clinical Data
Male
Comments

Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development RefNagy 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. They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras RefWood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. 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 RefNagy 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. 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.

*Current ATCC stocks of R1 cells are beyond passage 14. Current stocks of alternative subclone of R1 cells, designated R1/E (ATCC SCRC-1036), are below passage 14 and have been shown to be germline competent.


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.
Complete Growth Medium
The base medium for this cell line is ATCC-formulated ES-DMEM (ATCC® SCRR-2010). To make the complete growth medium, add the following components to the base medium: 2.0 mM L-Alanyl-L-Glutamine (ATCC® 30-2115) 0.1 mM 2-mercaptoethanol (Invitrogen Cat. No. 21985) 1X MEM Non-essential Amino Acid Solution (ATCC® 30-2116) 1,000 U/ml mouse leukemia inhibitory factor (LIF) (Chemicon Cat. No. ESG1107) 15% FBS, ES Cell Qualified (ATCC® SCRR-30-2020) Complete ES-DMEM is stable for 15 days when stored at 2°C to 8°C. This medium is formulated for use with a 5% CO2 in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO2 in air atmosphere is then recommended.)
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
Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.
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 RefNagy 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. They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras RefWood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. 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 RefNagy 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. 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.
*Current ATCC stocks of R1 cells are beyond passage 14. Current stocks of alternative subclone of R1 cells, designated R1/E (ATCC SCRC-1036), are below passage 14 and have been shown to be germline competent.
Year of Origin August, 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

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
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