Illustration of different steps for the acquisition, characterization and banking of parental CHO K1 stock cells. Yellow boxes illustrate sequential cell handling steps and dark blue boxes cell banking stages. Orange boxes indicate off-line testing and assessment steps.
Schematic illustration of the steps taken to delete the first allele of exon 6 on the GS gene in CHO K1 cells. Green boxes illustrate sequential cell handling steps and dark blue boxes cell banking stages. Orange boxes indicate off-line testing and assessment steps.
A full description of the targeting strategy is shown below.
(A) Illustration of rAAV/GS DNA construct to target deletion of exon 6 of the hamster GS gene. The GS left and right homologies correspond to genomic DNA sequences flanking exon 6 of the hamster GS gene, loxP sequences correspond to recognition sequences for the bacteriophage Cre recombinase enzyme
(B) Illustration of production of vector plasmid particles for CHO cell transduction.
(C) Deletion of chromosomal loci. Recombination between left and right homology regions in rAAV vector (A) and CHO chromosomal DNA (B) results in deletion of intervening chromosomal DNA (GS Exon 6) and insertion of Neor flanked by two loxP sites (C). Neor is then removed using the Cre/LoxP system.
Schematic illustration of steps to delete allele 2 of the GS gene. Pink boxes illustrate sequential cell handling steps and dark blue boxes cell banking stages. Orange boxes indicate off-line testing and assessment steps.
The same targeting strategy that was used to remove exon 6 from the first allele was used.
MOLECULAR BIOLOGICAL VALIDATION OF KNOCKOUT
Knockout of exon 6 was confirmed by PCR and sequencing.
FUNCTIONAL VALIDATION OF KNOCKOUT
When transferred to media lacking glutamine, the wild type CHO cells remain viable whilst the GS-/- cells are unable to survive. However, when transferred to media supplemented with 4mM glutamine, both cell lines grow at comparable rates.
A frozen vial of GS null HD-BIOP3 cells was transferred to LakePharma Inc. (CA, USA) for adaptation to suspension growth in chemically defined, animal component free media (this work was carried out between 4th August 2014 and 8th July 2015). Following transfer back to Horizon, the cells were sent to Wuxi Apptec for banking under GMP and testing for adventitious agents in line with ICH Q5.
Full details of this are available as part of the cell line history report.