Applied Microbiology and Biotechnology

, Volume 74, Issue 1, pp 152–159 | Cite as

Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin

Applied Genetics and Molecular Biotechnology
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Abstract

Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75–89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54–87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45–79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.

Keywords

Chinese hamster ovary (CHO) cells Lactate dehydrogenase-A (LDH-A) Small interfering RNA (siRNA) Specific lactate production rate 
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Notes

Acknowledgments

This research was supported in part by grants from the Ministry of Commerce, Industry, and Energy and Daejeon city (Bio/RIS program) and the Ministry of Education (Brain Korea 21 Program).

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Department of Biological SciencesKorea Advanced Institute of Science and TechnologyDaejeonSouth Korea

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