A novel approach for regulated trnasgene expression in mammalian cells

Abstract

A novel ‘screen and insert’ strategy to improve the ease, efficiency and reproducibility with which tightly regulated transgenes can be generated is tested in human cell lines. The strategy involves firstly identifying and characterising by flow cytometry those rare clones whose integration site allows optimal regulation of a reporter gene (encoding the Green Fluorescent Protein, GFP) by the tetracycline (Tet- OFF) inducible system. CVe-mediated site-specific recombination between two mutant loxP sites (lox71 and lox66) is then used to insert any gene of interest (for testing purposes the luciferase ORF was used) downstream of the tightly regulated promoter in such clones. To establish the ‘screen and insert’ approach in cell culture the human fibrosarcoma cell line, HT1080, and the telomerase immortalised retinal epithelial cell line, hTERT-RPEl, were used. The low frequency of Oe-mediated insertion (~1/105 of transfected cells) requires a method of selection for isolating clones and two methods are described. The first (System One) involves insertion of a gene of interest (GOI) which is linked to an IRES-hygromycin cassette to enable selection in hygromycin for the insertion event. A limitation of this strategy, however, is that the GOI must be expressed whilst selection takes place and this may be undesirable for certain GOIs (e.g. when a gene’s product is toxic). The second method (System Two) avoids this limitation by taking advantage of flp-mediated site-specific excision to delay GOI expression until selection for recombination is complete. The utility of the validated ‘screen and insert’ approach is demonstrated by use of the RAD52 gene (involved in homologous recombination and DNA repair) and the I-Scel endonuclease as GOIs. The resulting cell lines will be used to characterise the deleterious effects of RAD52 and I-Scel expression on genome stability and cell viability.