Faithful activation of an extra‐bright red fluorescent protein in “knock‐in” Cre‐reporter mice ideally suited for lineage tracing studies

Abstract

The considerable potential of Cre recombinase as a tool for in vivo fate-mapping studies depends on the availability of reliable reporter mice. By targeting a tandem-dimer red fluorescent protein (tdRFP) with advanced spectral and biological properties into the ubiquitously expressed ROSA26 locus of C57BL/6-ES cells, we have generated a novel inbred Cre-reporter mouse with several unique characteristics. We directly demonstrate the usefulness of our reporter strain in inter-crosses with a "universal Cre-deleter" strain and with mice expressing Cre recombinase in a T lineage-specific manner. Cytofluorometric and histological analyses illustrate: (i) non-toxicity and extraordinary brightness of the fluorescent reporter, allowing quantitative detection and purification of labeled cells with highest accuracy, (ii) reliable Cre-mediated activation of tdRFP from an antisense orientation relative to ROSA26 transcription, effectively excluding "leaky" reporter expression, (iii) absence of gene expression variegation effects, (iv) quantitative detection of tdRFP-expressing cells even in paraformaldehyde-fixed tissue sections, and (v) full compatibility with GFP/YFP-based fluorescent markers in multicolor experiments. Taken together, the data show that our C57BL/6-inbred reporter mice are ideally suited for sophisticated lineage-tracing experiments requiring sensitive and quantitative detection/purification of live Cre-expressing cells and their progeny. IntroductionCre recombinase is a small, bacteriophage P1-derived integrase that catalyses defined DNA recombination events between specific target sites, termed loxP (locus of crossover [x] in P1) [1]. LoxP sites are composed of two 13-bp inverted repeats and an asymmetric 8-bp spacer sequence, which endows individual loxP elements with a defined orientation [2]. The result of Cremediated recombination between two loxP sites de- pends on their specific orientation relative to one another: a DNA sequence flanked by two directly repeated loxP elements is cut out as a circular molecule, an effectively irreversible reaction due to loss of the excised product. In contrast, DNA flanked by two oppositely oriented sites will be inverted, a fully reversible process. Importantly, both activity and specificity of Cre recombinase are retained in eukaryotic cells [3], permitting the use of Cre as a genetic engineering tool in any cellular context. In combination with gene targeting techniques, the Cre/loxP recombination system has revolutionized the genetic analysis of mice [4,5]. Cre-mediated removal of loxP-flanked (floxed) selection cassettes, which are invariably introduced during the process of gene targeting into the mouse genome, and which may have unpredictable effects on neighboring genes, is already a routine manipulation. Also, conditional deletion of floxed genomic DNA sequences has become a rather standard approach for assessing tissue-, cell-type-or developmental stage-specific gene functions, provided appropriate Cre-transgenic mouse strains are available. Less common ...

The transcriptional coactivator Bob1 promotes the development of follicular T helper cells via Bcl6

“…ROSA/tdRFP mice (Luche et al , 2007), Pou2af1 −/− mice (Nielsen et al , 1996), and Pou2af1 −/− OTII mice were bred and maintained at the animal facility of the Max‐Delbrück‐Center. Mice were housed 5 mice/cage at a 12‐h day–night cycle with free access to tap water and food pellets.…”

αPIX RhoGEF Supports Positive Selection by Restraining Migration and Promoting Arrest of Thymocytes

“…Eight-to twelve-week-old littermates or age-matched mice were analyzed. For two-photon analysis, aPix 2 mice (C57BL/6) were crossed to histone-GFP mice (19) and rosa26 tdRFP mice (20). Expression of RFP was induced by pTa2Cre (20).…”

“…For two-photon analysis, aPix 2 mice (C57BL/6) were crossed to histone-GFP mice (19) and rosa26 tdRFP mice (20). Expression of RFP was induced by pTa2Cre (20). Git2 2/2 mice (21) on a C57BL/6 background were crossed to OT-II + mice, and heterozygous offspring were analyzed.…”

Suboptimal B‐cell antigen receptor signaling activity in vivo elicits germinal center counterselection mechanisms

“…tamoxifen administration into R26R-tdRFP;mb1cre-ER T2+ mice. A double administration of tamoxifen at a 4 days interval resulted in activation of tdRFP expression [18] in roughly 30% of the splenic B cells 5 days after the initial application (Fig. 2C).…”

“…3C and D). This indicates inefficient in vivo T-cell activation when Syk kinase activity is curtailed in professional APCs.To further address the resistance of GC B cells to delete Syk kinase, we mated Syk fl/Zap-70 ;mb1creER T2+ mice with R26R-tdRFP mice [18] to provide an independent genetic reporter for Cre activity and repeated the infection experiments according to the schedule outlined above (Fig. 2D).…”