This mouse ES cell line has been shown to be germline competent.xa0

The B6/BLU ES cell line was derived from a C57BL/6 transgenic line that contains a LacZ reporter. The transgene is a -globin LacZ fusion expressed exclusively in peripheral red blood cells. The transgene was assembled in pUC19, and contains the 1.9 kb KpnI-PvuII human 54 HS-2 fragment, upstream from the marked ß-globin transgene. The 5 part of the human ß-globin gene was fused to a 3 kb NcoI-BglII fragment obtained from pLacD. The 3 part of the -globin gene consisted of a 2.8 kb BamHI-XbaI fragment that contains the 3 end of exon 2, intron 2, exon 3 and 3 flanking sequence. 5 HS-2 was inserted in the genomic (5 to 3) orientation with respect to the transgene. The transgene was isolated from the plasmid vector backbone by cleavage with XhoI and SalI . Instead of determining chimerism visually by coat color or assessing chimerism genotypically in a tail DNA, chimerism is assessed quantitatively in the mesoderm by assessing the LacZ expression in a single drop of tail blood obtained at weaning.

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.

Hughes, DE et al. Genetic variation in C57BL/6 ES cell line. Mamm. Genome 18: 549-558, 2007. PubMed: 17828574

Ware, BC, et al. Utiltiy of a C57BL/6 ES line versus 129 ES lines for targeted mutations in mice. Transgenic Res. 12: 743-746, 2003. PubMed: 14713204

Graubert A T, et al. Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene. Nucleic Acids Research. 26 (12) :2849-2858, 1998. PubMed: 9611227