Choosing A Fluorescent Site Specific Recombinase Reporter Allele

Briefly, the choice of a fluorescent reporter should be considered carefully, with attention paid to its:      

A) brightness                                                                                                                                                                   B) folding / maturation time (a monomeric FP, such as superfolder GFP, will not behave like a oligmeric reporter, such as dsRED)                                                                                                                                                C) stability (both while "live" and after fixation, as some fluorescent proteins become dim/inactive following fixation).

Each of these criteria will each influence your interpretation of a Cre driver's activity/expression domain. Also, consider whether any antibodies are available to your reporter of interest for imaging, etc. before starting time consuming mouse crosses.

Another factor to consider is the nature/length of the stop cassette preceding the reporter. The more complex and longer it is, the more likely it will recombine less efficiently.

Finally, whether your reporter of interest utilizes the endogenous Rosa26 (or Hprt) promoter, or contains a synthetic promoter (such as CAGGS, CMV, B-actin, or Ubiquitin), and has a downstream stabilizing element (such as a WPRE), or destablizing sequence (PEST or degron), will all influence reporter activity.

For an example of different reporter activities, consider the image below using the older Rosa26-LSL-EYFP reporter and the newer Rosa26-ai6 (zsGreen) reporter. Note that the heart on the left is actually homozygous for the EYFP reporter, while the one on the right is heterozygous for the zsGreen reporter. These two hearts were photographed at the same time, at the same exposure, side by side one another.

Two hearts were isolated from embryos following crossing a transgenic Nxk2.5::Cre driver line to either A) a Rosa26-LSL-EYFP reporter (on the left) or B) a Rosa26-ai6 (zsGreen) reporter (on the right).

Two hearts were isolated from embryos following crossing a transgenic Nxk2.5::Cre driver line to either A) a Rosa26-LSL-EYFP reporter (on the left) or B) a Rosa26-ai6 (zsGreen) reporter (on the right).