title: Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.
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.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-Dong, Yuseong-Gu, Daejeon 305-701, South Korea.
authors.forename: Sung Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Gyun Min
authors.initials: GM
authors.lastname: Lee
mesh_terms: 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
chemical_list: D034741:RNA, Small Interfering; D019344:Lactic Acid; D013926:Thrombopoietin; D007770:L-Lactate Dehydrogenase