Nucleic Acids Research, 2012, Vol. 40, No. 10 4707 Figure 3. The cassette functions predictably in different gene contexts. (A) RT–PCR detection of spliced products for fLUC and abscissic acid 8’-hydroxylase (CYP707A3) harboring the P5SM cassette inserted within the E/R, K/R or E/S sequence contexts, upon induction with AtL5 (+) or LUC as a control ( ). (B) Relative amounts of SP-I transcript for fLUC constructs determined by RT–qPCR. Data are averages with number of biological replicates (n) and standard deviations shown. The p-value was determined using the paired t-test. (C) Luciferase activity in relative light units of fLUC constructs normalized to total protein upon induction with AtL5 (+) or control ( ). Fold induction by AtL5 is shown at the top of each bar. (D) RT–PCR detection of spliced products for PSY harboring the P5SM cassette inserted within the C/R sequence context upon induction with AtL5 (+) or LUC as a control ( ). A schematic representation of the identiﬁed aberrant spliced products (right) shows cryptic splicing from a proximal GU in the coding sequence (sequences are shown in Supplementary Figure S6). purine-rich A. thaliana sequence and tested a construct containing the hybrid P5SM sequence, eGFP-HyP5SM (Supplementary Figure S3). This construct exhibits strong induction ( 19-fold) by OsL5, almost no induction ( 1.5-fold) by AtL5, and very low background ﬂuores- cence (Figure 4A and C). With such low background ﬂuorescence, slight variations in this value resulted in a large standard deviation for fold induction between indi- vidual leaf samples. Furthermore, it was determined that DsRed2, the ﬂuor- escent protein used as a normalization standard in all of the experiments (see ‘Materials and Methods’ section), contributed to the residual background ﬂuorescence. In the absence of DsRed2, little to no background above autoﬂuorescence was observed for eGFP-HyP5SM in the absence of OsL5 induction (Supplementary Figure S4). With the exclusion of DsRed2 and with subtraction of autoﬂuorescence, both of which had not been done in previous experiments, it was revealed that the acti- vation of gene expression is very strong ( 97-fold on average) (Figure 4B). Correspondingly, western blot analysis with a GFP-speciﬁc antibody to measure protein levels detected the expressed protein only in OsL5-induced samples (Supplementary Figure S5). Consistent with the protein ﬂuorescence and immunoblot data, RT–PCR analysis revealed an almost complete switch in splicing upon induction by OsL5 but not AtL5 (Figure 4D). DISCUSSION Our construct designs and the associated bioinformatics estimates for genome coverage include two simplifying as- sumptions which impose conservative restrictions on gene context. First, we artiﬁcially limit the site of cassette in- sertion to within the ﬁrst half of ORFs, so that even in the absence of NMD mechanisms, the exon-retained spliced product would not generate functional protein. However, premature termination codons have been shown to activate NMD as long as they are upstream of an exon