Hypoxia Investigation
The ES Core has recently completed an investigation into the hypoxic growth conditions of both MEFS and the SCC10 ES line (129X1Sv/J). The ES Core initially analyzed hypoxic conditions on the growth of mouse embryonic feeder cells. Based on the robust growth and extended passage capability noticed in the MEF culture, the core began to condition the SCC10 ES cell line to hypoxic growth and evaluate their performance. The hypoxic growth and expansion of all mouse embryonic feeders and ES colonies was conducted in a humidified incubator with the following gas combination: 3% atmospheric oxygen; 95% nitrogen; 2% atmospheric carbon dioxide.
The hypoxic conditioned SCC 10 ES line was expanded four days in 3% oxygen, and chimeric mice were produced via standard blastocyst microinjection. Two chimeric founder males were bred to wild type C57BL/6 Taconic females, and both males have transmitted ES derived genetic markers through the germline (see Table 1).
Table 1
Mouse
#Sex Genotype D.O.B. Generation # Sired
F1s# Agouti
F1s% Germline
Transmission2 M chimera 3/27/07 N0 44 42 95% 3 M chimera 3/27/07 N0 24 5 21% There were no observed differences in morphology or growth rate of SCC 10 ES cells cultured under hypoxic conditions. In addition, hypoxically grown ES cells retain a 40XY karyotype. The ES Core is currently investigating possible strain-specific differences within hypoxic culture conditions by analyzing the B6/BLU ES line (C57BL/6 Taconic).
The ES Core routinely expands MEFs under hypoxic conditions, and will now expand all SCC10 clones for microinjection purposes under hypoxic conditions as well.
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