Abstract

In this study, the adequacy of β-galactosidase (β-gal) as marker for models that requires durable and high level gene expression in the absence of selective pressure was investigated. Chinese hamster ovary (CHO) cells were transfected with expression vector pcDNA4/HisMax-TOPO/lacZ containing lacZ and zeocin resistant genes. 26 recombinant CHO cell lines were established using lipid cationic transfection (TransFast™ Transfection Reagent) as DNA transfer method. 6 clones were productive in the expression of β-gal when grown in the presence of zeocin as the selective pressure. 2 sub-clones, TF8 (1) and TF9 (7) grown for 11 passages in nonselective medium which maintained high levels of protein expression with specific β-gal activity in the absence of zeocin were almost constant at 1.5704 and 4.3017 units β-gal activity/mg protein respectively. Specific growth rate of TF8 (1) and TF9 (7) in the absence of zeocin were approximately 0.638 and 0.656 day⁻¹ respectively. The expression of β-gal does not affect the cell growth and the transfectants were stable population in terms of cell viability. Removal of zeocin from the media increases specific growth rate from a range of 24 to 52% and β-gal protein production in reference to specific activity increases from 128 to 320% with the capability to be expanded to larger volumes. In this study, we demonstrate transfected CHO cells with the ability to produce β-gal without the presence of zeocin for at least 11 passages.

Key words: Chinese hamster ovary (CHO), β-galactosidase, clone stability, selective pressure

Abbreviation

 PEG, polyethylene glycol; SOD, superoxide dismutase; CAT,catalase; POX, peroxidase.