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. 2020 Aug 4;10:13125. doi: 10.1038/s41598-020-70022-0

Figure 5.

Figure 5

Investigation of potential cryptic splice sites within rtTA. (A) Predicted cryptic alternative splice sites within (top): original rtTA-M2, (middle): G72V-rtTA-M2 and (below): commercially codon optimised (cop)-rtTA-M2 using ASSP program. The default cut-off values of the ASSP program was used. The cut-off 2.2 for acceptor sites and 4.5 for donor sites have shown to correctly predict 75 to 80% of cryptic splice sites (Wang M and et al. 2006). (B) Removing the eight potential cryptic splice sites alone, or combination of all eight, improved the tightness of Tet-On system. (C) Comparison of the fold induction of pSBtet-5 with removing all cryptic splice sites in pSBtet-5. Combining G72V mutation with the eight cryptic splice sites removed, resulted in a non-responsiveness Tet-On system (D) Induction of luciferase expression in mutated rtTA-M2 proteins upon doxycycline induction. The missense E107Q mutation at position 320 bp showed lower induction. Experiments were carried out 96 h post-transfection. Statistical analysis: one-way ANOVA test with Bonferroni post-test correction (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).