Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice

• Published in: Disease models & mechanisms Vol. 4; no. 4; pp. 537 - 547
• Main Authors: Chen, Sara X, Osipovich, Anna B, Ustione, Alessandro, Potter, Leah A, Hipkens, Susan, Gangula, Rama, Yuan, Weiping, Piston, David W, Magnuson, Mark A
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Ltd 01.07.2011; The Company of Biologists Limited; The Company of Biologists
• Subjects: 3' Untranslated regions; 5' Untranslated Regions; Alleles; Animals; Brightness; Chromosomes, Artificial, Bacterial - genetics; Cloning; Copy number; Embryo cells; Embryo, Mammalian - metabolism; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Fluorescence; Gene expression; Genetic Loci - genetics; Genetic Vectors - genetics; Luminescent Proteins - metabolism; Mice; Mutagenesis, Insertional - methods; Organ Specificity; Plasmids; Polyadenylation; Proteins - genetics; Rabbits; Recombinase; Recombinases - metabolism; Resource; RNA, Untranslated; Stem cells; Transgenes; Translation
• ISSN: 1754-8403; 1754-8411
• DOI: 10.1242/dmm.006569

Fluorescent 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.