Fig. 3

Promoter RegCG has a transcription start site in the intron I. The transcription start site (TSS) from RegCG in the chimeric promoter RegCG/GRE/CMV was analyzed in cells derived from CHO DG44 cells co-transfected with pRegCG/GRE/CMV-aTNF-L and pRegCG/GRE/CMV-aTNF-H vectors. Total RNA was isolated from a culture of cells in the exponential phase, and cDNA was prepared. Primers that anneal to different parts of RegCG and cytomegalovirus (CMV) were used. a) Schematic representation of the recombinant anti-TNF-L gene commanded by the RegCG/GRE/CMV promoter, and the endogenous ACTB gene. The dotted lines indicate the region of the corresponding β-actin gene with the RegCG portion of the recombinant promoter. The lower arrows indicate the position and direction of the primers, where F and R indicate the forward and reverse primers, respectively. The upper arrows indicate the position of the theoretical TSS. b and c RT-PCR analysis of the recombinant promoter RegCG/GRE/CMV. At the bottom of the figure, RNA (total RNA), vector (plasmid DNA of pRegCG/GRE/CMV-aTNF-L vector), and primers are indicated. The primers used are indicated with numbers corresponding with numbering in A. This assay is representative of two experiments. d RT-PCR analysis of the β-actin gene