The color of mice: in the light of GFP-variant reporters

The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ, the bacterial beta-galactosidase gene has been the marker of choice for most studies in the mouse. However, over the past 3 years another valuable reporter has emerged, and its attractiveness is reflected by an explosion in its use in mice. Green fluorescent protein (GFP), a novel autofluorescent genetic reporter derived from the bioluminescent jellyfish Aequorea victoria, currently represents a unique alternative to other gene-based reporters in that its visualization is non-invasive and so can be monitored in real-time in vitro or in vivo. It has the added advantage that it can be quantified by, for example, flow cytometry, confocal microscopy, and fluorometric assays. Several mutants of the original wild-type GFP gene that improve thermostability and fluorescence have been engineered. Enhanced GFP is one such variant, which has gained popularity for use in transgenic or targeted mice. Moreover, various GFP spectral variants have also been developed, and two of these novel color variants, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), can also be used in mice. Since the spectral profiles of the ECFP and EYFP color variants are distinct and non-overlapping, these two reporters can be co-visualized, and are therefore ideal for in vivo double-labeling or fluorescent energy transfer analyses. The use of GFP and its color variants as reporters provides an unprecedented level of sophistication and represents the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal.     

Optimized expression of dual reporter genes in transient transfection of purified Toxoplasma gondii using different promoters

Fluorescent protein and luciferase genes are valuable reporter genes and have been widely used for noninvasive monitoring of gene expression in living tissues and cells. We tested expression of the dual reporter genes in transient transfection of purified Toxoplasma gondii tachyzoites. Two copies of the enhanced yellow fluorescent protein (EYFP) gene were put under the control of 3 representative T.?gondii promoters (GRA1, SAG1, and DHFR). Fluorescence from each EYFP reporter was significantly higher than that from a green fluorescent protein (GFP) reporter. The GRA1-EYFP reporter gave the highest fluorescence. Although both fluorescence and luciferase were expressed in the dual reporter system, the luciferase reporter was more efficient than either the EYFP or GFP reporters, and it required fewer parasites to be successfully used.     

Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal

Background  

Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP) from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes [1]. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP) in embryonic stem (ES) cells and mice [2].     

R26R-GR: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.     

Development of Gateway Binary Vectors,R4L1pGWBs,for Promoter Analysis in Higher Plants

We developed a new series of Gateway binary vectors for plant transformation, R4L1pGWBs, which allow easy construction of promoter:reporter clones. R4L1pGWBs contain a recombination attR4-attL1-reporter cassette, and thus an attL4-promoter-attR1 entry clone was efficiently incorporated by the Gateway LR reaction, resulting in the generation of an attB4-promoter-attB1-reporter construct. The reporters employed in R4L1pGWBs were β-glucuronidase (GUS), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), G3 green fluorescent protein (G3GFP), G3GFP-GUS, and tag red fluorescent protein (TagRFP).     

Fluorescent protein vector for directional selection of PCR clones

Green fluorescent protein (GFP) has become a convenient and versatile tool as a reporter protein in many aspects of science. Here, we show that the enhanced yellow fluorescent protein (EYFP) variant may be used advantageously as a reporter system for directional cloning of blunt-ended PCR products. We have constructed a pUC18-derived plasmid containing a reporter gene coding EYFP cloned into the BamHI/HindIII sites. The blunt-ended PCR product is cloned into the SmaI site of that plasmid. A reverse PCR primer must be designed with extra bases on the 5' end that are required to introduce a ribosome binding site (rbs) for EYFP expression. The reporter gene coding EYFP is not expressed unless an rbs is introduced in the proper orientation at the 3' end of the cloned PCR insert. The results of this cloning procedure may be analyzed by simple visual inspection using a transilluminator. In most cases, successful directional cloning results in white fluorescent colonies. The proposed procedure is a convenient method that can reduce the time- and labor-intensive analysis of the clones obtained during blunt-ended PCR product cloning.     

Gene-trap-based target site for cre-mediated transgenic insertion

There is an increasing need for tissue-specific gene expression regulatory elements to study normal and disease development in the mouse. However, the cloning and characterization of these elements are time-consuming and costly. Thus, there is a particular need to be able to identify gene expression patterns without having to clone the promoter elements. Gene-trap strategies identify expression patterns assigned for endogenous genes using reporters, such as LacZ (Gossler et al., 1989; Skarnes, 1990) or green fluorescent protein (GFP) (Ishida and Leder, 1999; Zheng and Hughes, 1999). The gene-trap vector randomly inserts into the genome and "steals" regulatory elements for the reporter. Here we describe an improved gene-trap strategy, which allows an efficient Cre recombinase-mediated insertion of any transgene into the trapped loci as a post-integrational modification and links the expression of the transgene to that of the reporter.     

Screening of novel nuclear receptor agonists by a convenient reporter gene assay system using green fluorescent protein derivatives.

Nuclear receptors represent a very good family of protein targets for the prevention and treatment of diverse diseases. In this study, we screened natural compounds and their derivatives, and discovered ligands for the retinoic acid receptors (RARs) and the farnesoid X receptor (FXR). In the reporter assay systems of nuclear receptors presented here, two fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), were used for detection of a ligand-based induction and as an internal control, respectively. By optimizing the conditions (e.g., of hormone response elements and promoter genes for reporter plasmids), we established a battery of assay systems for ligands of RARs, retinoid X receptor (RXR) and FXR. The screening using the reporter assay system can be carried out without the addition of co-factors or substrates. As a result of screening of more than 140 compounds, several compounds were detected which activate RARs and/or FXR. Caffeic acid phenylethyl ester (CAPE), known as a component of propolis from honeybee hives, and other derivatives of caffeic acid up-regulated the expression of reporter gene for RARs. Grifolin and ginkgolic acids, which are non-steroidal skeleton compounds purified from mushroom or ginkgo leaves, up-regulated the expression of the reporter gene for FXR.     

The IRG mouse: a two-color fluorescent reporter for assessing Cre-mediated recombination and imaging complex cellular relationships in situ

The Cre-loxP system is widely used for making conditional alterations to the mouse genome. Cre-mediated recombination is frequently monitored using reporter lines in which Cre expression activates a reporter gene driven by a ubiquitous promoter. Given the distinct advantages of fluorescent reporters, we developed a transgenic reporter line, termed IRG, in which DsRed-Express, a red fluorescent protein (RFP) is expressed ubiquitously prior to Cre-mediated recombination and an enhanced green fluorescent protein (EGFP) following recombination. Besides their utility for monitoring Cre-mediated recombination, we show that in IRG mice red and green native fluorescence can be imaged simultaneously in thick tissue sections by confocal microscopy allowing for complex reconstructions to be created that are suitable for analysis of neuronal morphologies as well as neurovascular interactions in brain. IRG mice should provide a versatile tool for analyzing complex cellular relationships in both neural and nonneural tissues.     

Expression and recombination of the EGFP and EYFP genes in lentiviral vectors carrying two heterologous promoters

BACKGROUND: Expressing two genes in the progeny of stem and progenitor cells that are transduced with a unique viral vector is desirable in certain situations. We tested the ability of two lentiviral vectors to transduce human cells of hematopoietic origin and concomitantly express two reporter genes, either EGFP (enhanced green fluorescent protein) and DsRed2, or EGFP and EYFP (enhanced yellow fluorescent protein), from two internal promoters. METHODS: The vectors were generated from the pTRIP deltaU3 EF1alpha EGFP lentiviral vector. Following transduction of hematopoietic and non-hematopoietic cell lines, we performed FACS, PCR and Southern blot analyzes to quantify transduction, integration efficiencies and size of integrated lentiviral vectors, respectively. RESULTS: The detection of DsRed2 fluorescence appeared unexpectedly low in human cells of hematopoietic origin. Alternatively, a modification in the flow cytometry assay allowed us to distinguish between the two overlapping fluorescence signals emitted by EGFP and EYFP, when transduced cells were excited with a 488-nm laser beam. However, the low frequency of double-positive EGFP+ EYFP+ cells, and the existence of single-positive, mostly EGFP- EYFP+, cells, prompted us to search for recombinations in the vector sequence. Southern blotting of DNA obtained from transduced cells indeed demonstrated that recombination had occurred between the two closely related EGFP and EYFP sequences. DISCUSSION: These observations suggest that recombination occurred within the EGFP and EYFP genes, which differ by only four amino acids. We conclude that the insertion of two highly homologous sequences into a lentiviral backbone can favor recombination.     

Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.     

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.     

Temporally controlled site-specific mutagenesis in the germ cell lineage of the mouse testis

We have obtained a PrP-Cre-ER(T) transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER(T) recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER(T) is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER(T) line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis.     

On murine mammary epithelial stem cells: discovery, function, and current status

Cell-surface markers expressed on mammary stem cells and progenitors have helped to establish a preliminary mammary cell lineage hierarchy. Further characterization of these cells depends on overcoming several technical obstacles.Remarkable progress has been made in the past decade in the isolation and characterization of mouse mammary stem cells and progenitors, as nicely reviewed in the article by Visvader and Smith (2011). Following in the footsteps of the hematopoietic system and analogous to bone marrow transplantation, the mammary gland can be reconstituted following transplantation of cells into the cleared mammary fat pad (see review by Medina 2011). Taking advantage of these similarities as well as the availability of genetically engineered mice (GEM), our laboratory initially used magnetic bead and fluorescence-activated cell sorting (FACS) and SCA enhanced green fluorescent protein (EGFP) knock-in mice to identify mammary gland progenitors (Welm et al. 2002). We also attempted to identify and isolate quiescent cells using a BrdU label retention strategy that had been successfully applied in the epidermal and intestinal epithelium. Subsequently, the identification of several cell-surface markers expressed on mammary stem cells and progenitors has resulted in an explosion in the field, and helped to define a preliminary mammary cell lineage hierarchy. These studies on the normal mammary gland have also provided the basis for hypotheses into potential mechanisms accounting for the heterogeneity of breast cancer subtypes (Behbod and Rosen 2004).One intrinsic difference between the hematopoietic system and the mammary gland, however, is the requirement for tissue dissociation in the latter case to facilitate the isolation of single cells required for FACS sorting. Even when using freshly isolated cells, there is a concern that these rather lengthy dissociation protocols may alter the expression of cell-surface molecules and properties of cells following disruption of the mammary gland architecture. Even short-term cell culture of primary mammary epithelial cells may alter the expression of cell-surface molecules. At present, single gene markers of mammary stem cells have not been identified, so the application of knock-in mice, e.g., the use of LGR5-EGFP to identify intestinal stem cells and perform lineage-tracing experiments (Barker et al. 2007), has not been feasible. One alternative approach may be to use pathway reporters, as recently described by Zeng and Nusse (2010), who used an axin-lacZ knock-in mouse to identify cells with canonical Wnt signaling with increased mammary repopulating activity. We have used a similar approach in a p53-null mouse mammary cancer model following lentiviral transduction with a Wnt reporter construct to identify cells with enhanced canonical Wnt signaling. These cells displayed a significant overlap with cell-surface markers in the basal-like tumors shown to enrich for tumor-initiating cells (Zhang et al. 2010).The use of multiple pathway reporters with different fluorescent reporters may provide a new approach to complement the current dependence on cell-surface markers. Fluorescent reporters also have the potential to help precisely visualize and model the location of mammary stem cells and progenitors in situ using multiphoton and other sophisticated microscopic techniques. The ability to visualize single stem cells in their niche environment and to follow both symmetric versus asymmetric division ultimately will be required for the next advances in the field. Recent studies on the paracrine effects of the steroid hormones, estrogen and progesterone, on mammary gland stem cells and progenitors illustrate the need to understand the spatial relationships among the various epithelial and stromal cell types present in the mammary gland. These studies will need to include cells from the immune system such as macrophages, neutrophils, etc., and derivatives of mesenchymal stem cells. Hopefully, in the near future it may be feasible to reconstitute and study these interactions in vitro, but for the present time this can be studied in GEM models. In addition, there is increasing evidence for the coexistence of quiescent and active adult stem cells in mammals (Li and Clevers 2010), but these distinct populations and their spatial and temporal relationships in the mammary gland remain to be discovered. Application of single-cell analysis using newly developed microfluidic platforms has the potential to help elucidate the potential heterogeneity of signaling pathways and gene expression in mammary stem cells and progenitors. Finally, there is a critical need for lineage-tracing experiments in the normal mammary gland to validate the proposed hierarchy for stem cells and progenitors, as well as to identify the cells of origin for different subtypes of breast cancer. Comparative studies of the murine and human stem cell populations in both the normal mammary gland and different breast cancer subtypes hold enormous potential for the future. Thus, despite the remarkable progress in this field, much remains to be done.     

Genetic and spectrally distinct <Emphasis Type="Italic">in vivo</Emphasis>imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein

Background  

DsRed the red fluorescent protein (RFP) isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP) for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems.     

Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts.

To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters.