Figure 1.
(A) Sequence comparison of the human U6 promoter and the modified U6 promoter with the bacterial tetracycline operon (tetO). The modified portion is indicated in bold. (B–E) Schematic illustrations of the vector construction process. (B) The U6 promoter sequence with an SphI recognition sequence (GCATG|C) was ligated into a TA cloning vector. (C) To preserve the first transcribed nucleotide as guanosine, the plasmid was digested with SphI and blunted with Klenow fragment. (D) To avoid hairpin formation of integrated sequences (3), DNA oligomers were ligated in two separate ligation reactions. Oligo1 was ligated following AatII digestion. (E) Oligo2 including the transcriptional termination signal T5 was ligated following AatII and ApaI digestion. (F–J) Schematic maps of vectors used in this study. (F) U6pro/DNMT1. DNMT1-targeting hairpin siRNA is driven by the human U6 promoter. (G) U6pro/Blank. As a control, the transcriptional termination signal T5 was ligated immediately downstream of the human U6 promoter. (H) U6pro/tetO/DNMT1. DNMT1-targeting hairpin siRNA is driven by the modified U6 promoter regulated by tetracycline. (I) U6pro/tetO/DNMT1/Hygro. U6pro/tetO/DNMT1 with the hygromycin resistance gene. (J) tetR/Neo. The tetR coding region was ligated in an expression vector with the neomycin resistance gene.