Background
Fluorescent proteins (FPs) are widely used to monitor gene expression and study cellular dynamics in cells and tissues. However, many variables can significantly affect experimental outcomes when FPs are expressed in the context of transgenes or gene targeting vectors in cell lines and mice. Variables that are intrinsic to the FP itself include the excitation and emission wavelengths, as well as the quantum yield of the FP. Variables extrinsic to the FP include the transcriptional activity of the specific promoter used to drive expression of the FP, the translational efficiency of the mRNA generated and the stability of the mRNA. Although many of the intrinsic factors have been precisely characterized, the extrinsic factors have been more difficult to accurately assess. Specifically, the variability that occurs owing to transgene copy number and insertion site has been a major impediment in quantifying the genetic factors that influence FP reporter gene expression in cells and mice.
Results
The authors of this study overcome this difficulty by using recombinase-mediated cassette exchange (RMCE) to generate an allelic series in mouse embryonic stem cells (mESCs), thereby allowing the quantitative assessment of intrinsic and extrinsic variables that influence FP expression in cells and mice. Eight different ROSA26 alleles are generated and compared to one another to assess the effects of intrinsic and extrinsic variables on FP protein expression in cells, as well as the relative brightness of different color variants of FPs. The authors show that the combination of genetic elements in an expression cassette influences FP expression up to threefold, and that relative expressed brightness of individual FPs varied up to tenfold in relation to one another. Among the five FPs examined, Citrine was the brightest, followed by Apple, eGFP, Cerulean and finally Cherry. In vivo expression of Cherry in ROSA26Cherry mice was observed in all organs, yet varied up to 30-fold between different tissues.
Implications and future directions
These results should help investigators to make more informed choices regarding the design of transgenes or vectors for expressing FPs and other proteins in cells and mice. In addition, the basal plasmids used in this study have design features that enable their use in BAC recombineering strategies and that simplify other aspects related to the design of gene targeting and RMCE vectors. Overall, these resources will simplify the generation of new loxed cassette acceptor (LCA) alleles, gene targeting and RMCE vectors, and mouse models, especially those containing FPs.
