We did Lentiviral transfections of Ntera2 cells using a tet-inducible GFP/miRxy vector.
After tet induction cells were nicely green within approx. 12 h after induction. And now the problem: We cannot detect an increase of miRxy expression in these cells (using qRT-PCR; established for the specific miRNAxy), although they are green, meaning that the induction works, per se. Does anyone here have experience with tet-inducible miRNA expression vectors? Many thanks in advance!
Dear Dr. Silke, I guess you have cloned your target pre-microRNA into the vector. If that`s the case, I think, some unknown protein is inhibiting the processing of pre-miRNA transcript into mature miRNA. For example, you know LIN28 inhibits the Dicer processing of pre-let-7 into mature Let-7 (Rybak et al.Nat Cell Biol. 2008 ). In that case, its better to clone mature form.
Another possibility might be the incubation time. I didn`t get enough expression of my tet-inducible miRNA within 48 hrs, I got it in 72 hrs.
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