Figure 6

β-glucuronidase hairpin RNA ( hpGus )-derived small interfering RNAs (siRNAs) can mediate target transcript downregulation. (A) Strain S5, which contains the hpGus transgene and produces siRNAs (see Figure 3), was super-transformed with the Gus transgene to obtain S5:Gus lines W1 to W8. Relative Gus activity was determined by MUG assay (top panel). The mean of at least two independent biological replicates is shown with error bars representing the standard deviation. The second panel shows RNA blot analysis of 15 μg of total RNA hybridized with a full length Gus probe to detect small RNAs. The U6 transcripts are shown as loading control. Expression levels of the Gus and hpGus transgenes are shown in the fourth panel. Total RNA (10 μg) was hybridized with a full length Gus probe, detecting the1.8 kb transcript derived from the Gus transgene and the 1.1kb fragments corresponding to the single stranded loop region of the hpGus transcript. Ethidium bromide-stained ribosomal RNA bands are shown as loading control. DNA blot analysis was performed to determine transgene integrity (bottom panel). Genomic DNA was restricted with EcoRI and SalI, and hybridized with a full length Gus probe. Restriction fragments corresponding to the hpGus (2.7 kb) and Gus transgenes (3.2 kb) are present in most lines, indicating that both transgenes remain intact. (B) Secondary siRNAs are produced in some of these lines. Total RNA (15 μg) from S5:Gus lines W1 to W8 were resolved on 17% polyacrylamide and hybridized with a probe specific for the unique region that is present only in the Gus but not the hpGus transcript (see Figure 1 and Methods for details). Low levels of Gus-specific siRNAs were identified in lines W5 to W7. The U6 transcript is shown as loading control.