Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.
Mol Biotechnol
; 56(9): 833-8, 2014 Sep.
Article
en En
| MEDLINE
| ID: mdl-24841241
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Serina-Treonina Quinasas
/
Lactato Deshidrogenasas
/
Genes Letales
Límite:
Animals
Idioma:
En
Revista:
Mol Biotechnol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Año:
2014
Tipo del documento:
Article
País de afiliación:
Estados Unidos