Figure 3.
Confirmation of RNAi mutants. (A) Methylene blue staining of viable cells showing the drug resistance of a putative RNAi mutant as compared to the reporter cell line, 59Z4. (B) A Southern blot of EcoRI digested DNA using a probe unique to the gene trap identifies distinct gene-trap integration events by size. (C) Western and northern blots show that Hprt protein and RNA expression returns to levels similar to the parental cell line, PGG5-4. For the western blot, β-tubulin is shown as a loading control. For the northern blot, the 28S and 18S rRNAs are shown as a loading control. (D) A luciferase assay was used to confirm that HATR clones are RNAi-defective. The fold repression of F-luc by the luc1-shRNA is shown for the reporter (59Z4) and mutant cell lines. The data shown are the average of six independent luciferase assays, and error bars represent the standard error of the mean. The F-luc data was normalized to R-luc and the fold repression of F-luc by the control shRNA was set to 1 (not plotted). The reporter cell line represses the F-luc reporter 35-fold while the mutants repress 1- to 2-fold.