Transcriptional Activity Assessment of Two Different Eukaryotic Promoters In Vitro and In Vivo

Abstract: As a key element of a plasmid， the promoter determines the transcriptional activity of the exogenous genes. In order to screen for the effective promoter inside plasmid used in studying gene function， the transcriptional activity of two commonly used promoters， CMV and CAG were compared in vitro and in vivo by the fluorescence intensity of enhanced green fluorescent protein (EGFP). Firstly， the target gene N-cadherin (N-cad) was cloned into the plasmid of pCAG-MCS-EGFP， and confirmed by colony PCR， double digestion and sequence. Subsequently， these two plasmids (pEGFP-N-cad and pCAG-N-cad-EGFP) were transfected into HEK293T in vitro by calcium phosphate coprecipitation technique， and imaged under a fluorescence microscope， then the expression of N-cad was confirmed by Western blot and RT-PCR. Thirdly， the two plasmids were transfected into chicken embryonic spinal cord by electroporation， and imaged under a stereo fluorescence microscope. Finally， the EGFP positive spinal cords were selected and confirmed by frozen section under a fluorescence microscope， Western blot and RT-PCR. The results showed that these two promoters had equivalent transcriptional activity in vitro， but the artificially designed CAG promoter could effectively drive target gene expression in vivo when compared to the CMV promoter that originated from cytomegalovirus.

CSTR: 32200.14.cjcb.2015.10.0004