Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.
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.
Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.
Shirley S M
SS
Yip
Meixia
M
Zhou
John
J
Joly
Bradley
B
Snedecor
Amy
A
Shen
Yongping
Y
Crawford
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D003412:Cricetulus; D055786:Gene Knockout Techniques; D005804:Genes, Lethal; D045022:Lactate Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D019289:Pyruvic Acid / Q000378:metabolism
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D019289:Pyruvic Acid; D045022:Lactate Dehydrogenases; D017346:Protein Serine-Threonine Kinases