Considerations on the design of a transgene - (Feb/20/2014 )

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?

Thanks,

1) I am in the opinion that you should use insulators. They  do make a difference.

As both genes are close to each other, flanking both genes individually is not require. It is good enough to flank both genes as a unit, ie have one insulator 5' of gene 1 and a second insulator 3' of gene 2 (insulator -gene1-gene2-insulator)

To the best of my knowledge there is no gold standard insulator. Insulators do show species specific and even tissue specific behavior. However I would go with tDNA insulators, cause it seems quite good at protecting genes, relatively small (500bp) and one would guess that most cell types will have the proteins to recognize tDNA insulators. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771377/

2. Cannot say. How much expression do you need? But when in doubt I would go with CAG promoter. But do note that the CAGpromoter has some tissue specific behavior. It is not always the strongest promoter every instance. Do you have any other promoters?

3. A minor point, please note the tet inducible promoters do leak. So if you want to be extra safe you can either add tetR-Krab (transcriptional suppressor) fusion to the mix, or some selection marker to your gene structure that allows you select for cell which carry your cassette.. A system to select for the lost of your cassette would also be useful. HyTK is a nice fusion gene. Position selection on hygromycin and counterselection with ganciclovir.