I'm attempting to design a transgene for a knock-in recombination into a murine cell line genome. In general, I have 2 separate transgenes (i.e. 2 separate promotes) that I ultimately want to be able to cre/lox out. My first gene contains 3 separate coding sequences, separated by P2A sequences, under a CAG (or maybe minimal CMV promoter) promoter; one of those coding sequences is rtTA (for TetON system). The next gene, right downstream, will be under a tet-inducible promoter and will drive cre-recombinase expression. This will all be flanked with loxp sites and 1kb homologous arms. This transgene will be recombined into the typical rosa26 locus.
1) will I need to put insulators surrounding both transgenes? If so, is there a insulator sequence that's the "gold standard"?
2) Is a minimal CMV promoter sufficient to drive expression or should I use CAG (for the first transgene)?
3) Are there any glaring concerns I over looked in my transgene design?
Edited by Ahrenhase, 20 February 2014 - 08:59 AM.