Neha Dhami1, Drupad K Trivedi2, Royston Goodacre2, David Mainwaring3,4, David P Humphreys3. 1. Protein Sciences, UCB, 216 Bath Road, Slough, Berkshire, SL1 3WE, UK. neha.dhami@ucb.com. 2. Manchester Institute of Biotechnology and School of Chemistry, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. 3. Protein Sciences, UCB, 216 Bath Road, Slough, Berkshire, SL1 3WE, UK. 4. Pall Europe Limited, 5 Harbourgate Business Park, Southampton Road, Portsmouth, Hampshire, PO6 4BQ, UK.
Abstract
INTRODUCTION: Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production. OBJECTIVES: The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity. METHODS: To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi. RESULTS: Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production. CONCLUSION: Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.
INTRODUCTION:Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production. OBJECTIVES: The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity. METHODS: To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi. RESULTS: Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production. CONCLUSION: Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.
Entities:
Keywords:
CHO cells; Glucose metabolism; Oxidative stress; TCA cycle; m-Aconitase
Authors: Warwick B Dunn; David Broadhurst; Paul Begley; Eva Zelena; Sue Francis-McIntyre; Nadine Anderson; Marie Brown; Joshau D Knowles; Antony Halsall; John N Haselden; Andrew W Nicholls; Ian D Wilson; Douglas B Kell; Royston Goodacre Journal: Nat Protoc Date: 2011-06-30 Impact factor: 13.491
Authors: S C G Brezinsky; G G Chiang; A Szilvasi; S Mohan; R I Shapiro; A MacLean; W Sisk; G Thill Journal: J Immunol Methods Date: 2003-06-01 Impact factor: 2.303