Monitoring transfected cells without selection agents by using the dual-cassette expression EGFP vectors

Abstract

Conventional methods of selecting gene transfected cells by toxic agents may yield ambiguous results.It is difficult to determine whether cell death is due to selection agents or gene transfection, owing to the substantial overlap of the time-courses for both effects. Therefore, to determine transfection-induced cell toxicity, the mammalian expression vector pEGFP-N1 (CLONTECH Lab., Palo Alto, CA, USA) has been modified to the dual-cassette expression vectors named pEGFP-Ks by the relocation of its EGFP expression cassette. We have precisely monitored the cells transfected with this vector on our custom culture dishes, thereby bypassing the need for selection agent or fluorescent cell sorting. This is a useful method to screen genes encoding potential toxic or useful proteins without performing undesirable selection agent and also can be used to monitor the transfected cells for various purposes, either the inhibition or proliferation of mammalian cells for applications in biotechnology.

Transgenic resources for functional studies on the hsrω gene in Drosophila

“…To achieve this three PCR amplified DNA fragments were ligated. Two fragments were amplified from the hsrω gene and the third (EGFP fragment) from the pEGFP-N1 plasmid (Clontech; Kang et al, 2001). The first hsrω gene fragment was amplified from TSS2 to 3' end of ORFω (excluding its stop codon) with primers 1F and 1R (Table 2) with EcoRI and SpeI overhang sites, respectively.…”