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Fluorescent protein (FP) reporter alleles are useful both for identifying and purifying specific cell populations in the mouse. Here, we report the generation of mouse embryonic stem cells that contain a pancreatic and duodenal homeobox 1 (Pdx1) loxed cassette acceptor (Pdx1(LCA)) allele and the use of recombinase-mediated cassette exchange to derive mice that contain a Pdx1(CFP) (Cerulean) reporter allele. Mice with this allele exhibited cyan fluorescence within the previously well-characterized Pdx1 expression domain in posterior foregut endoderm. Immunolabeling showed that endogenous Pdx1 was coexpressed with CFP at all time points examined. Furthermore, fluorescence-activated cell sorting was used to isolate CFP-positive cells from E11.5 and E18.5 embryonic tissues using both 405 and 445 nm lasers, although the latter resulted in a nearly 50-fold increase in emission intensity. The Pdx1(CFP) allele will enable the isolation of specific foregut endoderm and pancreatic cell populations, both alone and in combination with other FP reporter alleles.

SUMMARYFluorescent proteins (FPs) have great utility in identifying specific cell populations and in studying cellular dynamics in the mouse. To quantify the factors that determine both the expression and relative brightness of FPs in mouse embryonic stem cells (mESCs) and in mice, we generated eight different FP-expressing ROSA26 alleles using recombinase-mediated cassette exchange (RMCE). These alleles enabled us to analyze the effects on FP expression of a translational enhancer and different 3′-intronic and/or polyadenylation sequences, as well as the relative brightness of five different FPs, without the confounding position and copy number effects that are typically associated with randomly inserted transgenes. We found that the expression of a given FP can vary threefold or more depending on the genetic features present in the allele. The optimal FP expression cassette contained both a translational enhancer sequence in the 5′-untranslated region (UTR) and an intron-containing rabbit β-globin sequence within the 3′-UTR. The relative expressed brightness of individual FPs varied up to tenfold. Of the five different monomeric FPs tested, Citrine (YFP) was the brightest, followed by Apple, eGFP, Cerulean (CFP) and Cherry. Generation of a line of Cherry-expressing mice showed that there was a 30-fold variation of Cherry expression among different tissues and that there was a punctate expression pattern within cells of all tissues examined. This study should help investigators make better-informed design choices when expressing FPs in mESCs and mice.

SummaryExtracellular signals in development, physiology, homeostasis, and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for being able to systematically compare the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, BMPs, activin A, Wnts, or Notch, and that can be adapted to any pathway that acts via DNA elements.