I am trying to generate RKO cells with constitutively active nuclear B-catenin. I have transfected with two different plasmids: pCl-neo beta catenin S33Y (addgene plasmid #16519) and BcatMutS33/S37.T41/S45 (addgene plasmid # 24204).

The cells transfect well, exhibiting an initial transfection efficiency of 30-50%, and are readily selected with G418.

I have encountered two issues with generating a stable clone, however:

Over time (in a matter days to weeks), the mutant B-catenin expression disappears. This is likely due to methylation of the CMV promoter. The SV40 promoter, however, is spared, allowing the cells to survive continued G418 selection.

Additionally, neither mutant B-catenin construct appears to reliably result in elevated nuclear B-catenin localization as judged by ICC. Only a small fraction of all of the cells which do express the mutant protein also exhibit nuclear localization of the protein.

Does anyone have suggestions of alternative vectors or promoters that might avoid methylation in the hypermethylating environment of RKO cells? Additionally, does anyone have suggestions of an alternative B-catenin mutation or construct that will more reliably localize to the nucleus?

Thank you for any and all help!