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AB2.2; Mouse ES Item ID: ATCSCRC1023

Permits and Restrictions

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Organism Mus musculus, mouse
Tissue inner cell mass
Cell Type embryonic stem cell
Product Format frozen
Morphology Spherical colony
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Age embryo
Gender Male
Strain 129S5/SvEvBrd (formerly 129/SvEvBrd)
Applications

Differentiation: AB2.2 cells have been successfully differentiated into cardiomyocytes from embryoid bodies.

Gene knock-out: AB2.2 retain very high germline transmission rates after being genetically manipulated making them an excellent candidate for targeted-mutation and gene knock-out experimentation. They have been used to create knock-out mice to study the role of epidermal proteins in normal development.

Gene knock-down: AB2.2 cells have been used in RNA interference research aimed at understanding the role of various proteins in tissue-specific development and function

Recombineering: A unique, fully end-sequenced, 129Sv BAC library consisting of 84,507 bacterial artificial chromosomes has been generated from AB2.2 ES cell DNA. This BAC library, referred to as bMQ BAC (www.geneservice.co.uk), is a publicly available BAC resource that can be used for the rapid construction of targeting vectors using current recombineering techniques.
Storage Conditions liquid nitrogen vapor phase
Clinical Data Male
Comments The line has been used extensively for the creation of knockout mice. The HPRT negative mutation makes the line useful for chromosome engineering. This mouse ES cell line has 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 Complete Growth Medium for ES Cells: Grow ES cells in Mouse ES Cell Basal Medium (ATCC SCRR-2011) that has been supplemented with the following components: 0.1 mM 2-mercaptoethanol (Invitrogen Cat. No. 21985) 1,000 U/ml mouse leukemia inhibitory factor (LIF) (EMD Millipore Cat. No. ESG1107) 15% FBS, ES Cell Qualified (ATCC SCRR-30-2020)Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C. This medium is formulated for use with a 5% CO2 in air atmosphere.
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 Liquid nitrogen vapor phase
Culture Conditions Temperature: 37°C Growth condition: feeder cells required
Name of Depositor A Bradley
Year of Origin 1988
References

Wolcik SM, Longley MA, Roop DR. Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b. J. Cell Biol. 154: 619-630, 2001. PubMed: 11489919

Adams DJ, et al. A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction. Genomics 86(6): 753-758. 2005. [PubMed: 16257172]

Festing MF, et al. Revised nomenclature for strain 129 mice. Mamm. Genome. 10(8):836, 1999.[PubMed: 10430671]

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

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