Figure 2.

Evaluation of AMPM expression systems by comparison of the level of expression of AMPM and of silencing efficacy. A. Schematic representation of the AMPM expression vectors. To generate the pAMPM-FL-exon and pAMPM-FLintron, the AMPM-FL cassette was inserted into the 5′-UTR and intron of Hyg^r, respectively, in pVITRO1. Gray boxes in the pAMPM-FL-exon and pAMPM-FL-intron indicate the AMPMFL cassettes. EF1a, the promoter of the rat translation elongation factor 1a gene; Hyg^r, the hygromycin resistance gene; pA, the human elongation factor 1a polyadeylation signal. B. The expression of a mature artificial miRNA targeting firefly luciferase. The pAMPM-FL-exon and pAMPM-FL-intron vectors were transiently transfected into HeLa cells and the total RNAs were isolated. Northern blot analysis using a radiolabeled oligonucleotide probe detected a band around 22 nt in length corresponding to the antisense strand region of AMPM-FL. Transfer RNAs (tRNAs) served as a loading control. C. Silencing efficacy of exonic and intronic AMPM-FL. The pAMPM-FLexon, pAMPM-FL-intron or pVITRO1 vector was transfected with the pGL3-Control reporter vector and pRL-TK normalization vector into HeLa cells. Normalized luciferase activities were standardized relative to levels in lysates from cells transfected with the empty vector, pVITRO1. The values are means with S.E.M. (n=3). D. The influence of the insertion of the AMPM cassette on the expression of Hyg^r. The pAMPM-FLexon, pAMPM-FL-intron or pVITRO1 vector was transfected into HeLa cells and the total RNAs were isolated 48 hours after transfection. RT-PCR was carried out with primers to detect Hyg^r and GAPDH mRNA. GAPDH served as a control.