Horizon Discovery successfully engineers CHO cells using rAAV-mediated homologous recombination
Glutamine Synthetase (GS)-negative CHO cell lines could accelerate the development of cell lines within the bioproduction industry by acting as a valuable resource for stringent selection. Currently, selection based on GS is frequently compromised by the endogenous expression of the enzyme.
Scientists at Horizon Discovery, a leading provider of research tools to support the development of personalized medicines, have used simple-to-develop, recombinant adeno-associated virus (rAAV), single-stranded DNA vectors to achieve the targeted deletion of GS in CHO cells.
Joshua Kapp, Business Development Manager at Horizon Discovery said, “We succeeded in this first attempt at targeted gene manipulation in CHO cell lines using rAAV-mediated homologus recombination in under six weeks* with a targeting efficiency of 1.18%. The study demonstrates that Horizon Discovery’s rAAV genome editing technology is an efficient tool for the routine modification of these cells.”
After characterising the growth characteristics and amenability to transduction and transfection of clonal CHO cells a suitable clone was selected for targeting. Following the sequencing of the CHO GS gene a targeting vector was designed to be homologous to exons four, five, six and seven, to avoid off-target recombination with potential pseudo-genes.
Transfected CHO-K1 cells were selected using antibiotic and sequence analysis performed on knock-out clones confirmed the correct integration of the vector and subsequent knock out of exon six (first allele). Studies are ongoing to achieve a bi-allelic knockout.
To access the full poster and for more information see: http://www.horizondiscovery.com/downloads/library/poster-pi3k-mutation-2012.pdf