Fig. S1.

Efficiency of T-RNA removal by T6- and T7-shRNA in cultured cells and example of shRNA-vector EGFP cassette expression in transgenic embryos. (A) Efficiency of different T-shRNAs (T1–T7) was assessed by measuring activity of a chimeric Renilla luciferase reporter containing the full-length mouse T-transcript sequence fused to the 3′ end of the luciferase coding region compared with that of the firefly luciferase control. T-shRNAs were cloned into a recombined active version of the pSico vector (EGFP cassette removed) and were cotransfected into NIH 3T3 cells with the chimeric T-luciferase gene to screen for knockdown efficiencies. Empty vector (pSico) and a GFP-shRNA (pSicoGFP) were included as negative controls. Both T6- and T7-shRNAs (shown in A) achieved considerable knockdown of the T-luciferase chimera, averaging 89% for T6-shRNA and 84% for T7-shRNA. The bar graph represents averages from five independent experiments. Statistical significance (P) of difference in normalized luciferase activity was determined using Student’s two-tailed t test. Other shRNAs tested (Table S1) gave knockdown efficiencies ranging between 54% and 75%. (B) Ubiquitous expression of GFP cassette in transgenic pSico-shRNA embryos in the absence of Cre-mediated recombination.