BIOT 236 |
| We attempted to reduce lactate production during mammalian cell culture by reduction of LDH-A gene expression in CHO host cell line by small-hairpin RNA method of gene silencing. Vectors expressing LDH-A specific shRNA were transfected into CHO cells and potential LDH-A depleted clones were identified. Several LDH-A depleted clones had extended longevity (98 % viable at day 10 compared to 49% viable for control) and reached higher peak cell density (11 x 106 cells/ml as compared to 6 x 106 cells/ml for parental). The lactate formation was reduced by <66% during batch culture and its intracellular LDH-A enzyme activity was significantly decreased. Additionally, LDH-A mRNA levels (the sole functioning LDH gene in CHO) were reduced by <50% in LDH-A depleted lines as compared to the parental line. These results demonstrate that reduction of LDH levels can potentially improve cell culture processes by increasing viable cell densities and the length of the bioreactor run. |
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Poster Session
5:30 PM-7:30 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster
Division of Biochemical Technology |