Figure 3

Test of different translation termination sequences for transient expression in HEK293T cells. A number of pCMV vectors were generated containing the opal stop codon and the following one (pCMV2.1), five (pCMV2.2), or 14 nucleotides (pCMV2.5) derived from genomic human Ig sequence or adenine as “fourth” stop codon nucleotide (pCMV2.4). Moreover, we also tested vector constructs containing an ochre stop codon followed by an adenine (pCMV2.5) and pCMV2.2 variants where the 3' terminal lysine codon was deleted (pCMV3.2) or replaced by a BamHI cloning site encoding the amino acids GS instead (pCMV4.2). Transient productions were done in HEK293T cells and tested by human IgG/Fc capture ELISA 48 h after transfection. Mean production levels and standard deviations of three productions are given. Cells transfected with the YFP reporter gene are used as negative control for the ELISA. Statical analysis was performed by comparing each vector with the vector pCMV using a t-test. Significance is indicated (* … p < 0.05 and ** p < 0.01). ND not determined.