Abstract
Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3.
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Acknowledgments
We thank the Analytical Operations Department at Genentech for performing titer and product quality assay, DNA Sequencing Group for all the sequencing support, DNA/RNA Synthesis Group for synthesizing all the primers and probes used in this study, and Media Preparation Group for all of the media used in this study.
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12033_2014_9762_MOESM1_ESM.tif
shRNA mediated down-regulation of PDHK1, 2, and 3 in CHO cells resulted in reduced lactate production in a fed-batch production evaluation without any significant impact on cell growth. (a) Lactate level for the control and the shRNA clones in a 14-day fed-batch production assay. Control clones were transfected with a mock vector and shRNA clones were transfected with a vector containing shRNAs for PDHK1, 2, and 3. Clone shRNA 1 is the PDK8 selected for the knockout study. (b) Statistical analysis on day 14 lactate level for control and shRNA clones (p = 0.014). (c) Day 14 IVCC for control and shRNA clones. (d) Statistical analysis on day 14 IVCC for control and shRNA clones (p = 0.252). (TIFF 358 kb)
12033_2014_9762_MOESM2_ESM.tif
Karyotyping analysis for clones PDK8 (a) and 1B9 (b). Clone PDK8 was determined to be pseudo-diploid with the number of chromosomes per cell ranging from 18 to 22. Clone 1B9 had duplicated genome with the number of chromosomes per cell ranging from 35 to 40. Representative examples of 10 metaphases are shown here for each clone. (TIFF 1029 kb)
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Yip, S.S.M., Zhou, M., Joly, J. et al. Complete Knockout of the Lactate Dehydrogenase A Gene is Lethal in Pyruvate Dehydrogenase Kinase 1, 2, 3 Down-Regulated CHO Cells. Mol Biotechnol 56, 833–838 (2014). https://doi.org/10.1007/s12033-014-9762-0
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DOI: https://doi.org/10.1007/s12033-014-9762-0