We have established a mice model using the CRISPR/Cas9 technique to make a conditional gene overexpression model, and the Rosa26-knockin fragment consists of CAG promoter-cDNA-3xflag-IRES-EGFP ,and we have crossed this line with TPO-Cre mice, which limit the geneA overexpression in thyroid tissues. However, I haven't seen any signal when I did the anti-Flag Western Blot of anti-flag IHC ,which means the target gene may not express. And I'm sure about the effect of Cre mice, because I have seen the phenotype of another gene crossed with the same Cre.

I have done the Sanger seqencing of mice gene to make sure if there's any mutation or truncation in the knock-in fragment, and there're no problem.

Now I have no idea where the problem is…

Thanks for any advice!