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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.

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.

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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

Liu Y, et al. Sox17 is essential for the specification of cardiac mesoderm in embryonic stem cells. Proc Natl Acad Sci USA. 104(10): 3859-3864, 2007. PubMed: 17360443

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Simpson EM, et al. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16(1):19-27, 1997. PubMed: 9140391

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