Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.
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.
Kim
Sung Hyun
SH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-Dong, Yuseong-Gu, Daejeon 305-701, South Korea.
Lee
Gyun Min
GM
D000818:Animals; D001709:Biotechnology / Q000379:methods; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013926:Thrombopoietin / Q000096:biosynthesis*; D014162:Transfection
D034741:RNA, Small Interfering; D019344:Lactic Acid; D013926:Thrombopoietin; D007770:L-Lactate Dehydrogenase