Reporter Mouse Models Research Articles

Cytokine Reporter Mice: The Special Case of IL‐10

IL-10 is one of the key cytokines preventing inflammation-mediated tissue damage. In an attempt to identify IL-10-producing cells in vivo, several groups have recently developed IL-10 reporter mouse strains. Up until now, in total, eight IL-10 reporter strains have been published. This incomparable interest in IL-10 reporter mice emphasizes the importance and difficulties in tracking and subsequently investigating the role of IL-10-producing cells in infectious, inflammatory, autoimmune and cancer diseases. In this review, I summarize and compare the properties of those published IL-10 reporter mouse models. I also discuss the necessity to develop new strategies to generate 'multi-cytokine' reporter mouse models enabling highly sensitive in/ex vivo detection of many cytokines in the same single cell. Such 'multi-cytokine' reporter mice will enable to reconsider the dichotomy 'T-effector versus T-regulatory' paradigm and to provide an accurate revised model for cellular sources of cytokines. Finally, I propose to launch cooperative, international initiatives to promote and coordinate the generation of accurate, combinatorial, reporter mice for every individual murine cytokine.

Transgenic mouse analysis of Sry expression during the pre‐ and peri‐implantation stage

The SRY/Sry gene is expressed in pre-Sertoli cells of the male genital ridge and functions as the mammalian testis determining factor (TDF). In addition, expression of SRY/Sry outside the genital ridge has been reported, including preimplantation embryos, although the functional significance of this is not well understood. Using Cre-mediated lineage studies and transgenic reporter mouse models, we now show that promoter sequences of human, pig and mouse SRY drive robust reporter gene expression in epiblast cells of peri-implantation embryos between embryonic day (E) 4.5 and E6.5. Analysis of endogenous Sry expression revealed that linear transcripts are produced by means of multiple polyadenylation sites in E4.5 embryos. Within the epiblast, SRY reporter expression mimics the expression seen using a Gata4 reporter model, but is dissimilar to that seen using an Oct4 reporter model. In addition, we report that overexpression of mouse Sry in embryonic stem cells leads to down-regulation of the core pluripotency markers Sox2 and Nanog. We propose that SRY/Sry may function as a male-specific maturation factor in the peri-implantation mammalian embryo, providing a genetic mechanism to help explain the observation that male embryos are developmentally more advanced compared with female embryos, and suggesting a role for SRY beyond that of TDF.

Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

BackgroundReporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available.ResultsWe demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts.ConclusionThe three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

Memory CD4 T cells emerge from effector T-cell progenitors

A hallmark of adaptive immunity is the generation of memory T cells that confer long-lived, antigen-specific protection against repeat challenges by pathogens. Understanding the mechanisms by which memory T cells arise is important for rational vaccination strategies and improved therapeutic interventions for chronic infections and autoimmune disorders. The large clonal expansion of CD8 T cells in response to some infections has made the development of CD8 T-cell memory more amenable to study, giving rise to a model of memory cell differentiation in which a fraction of fully competent effector T cells transition into long-lived memory T cells. Delineation of CD4 T-cell memory development has proved more difficult as a result of limitations on tracking the smaller populations of CD4 effector T cells generated during a pathogenic challenge, complicating efforts to determine whether CD4 memory T cells are direct descendants of effector T cells or whether they develop by alternative pathways. Here, using two complementary cytokine reporter mouse models to identify interferon (IFN)-gamma-positive effector T cells and track their fate, we show that the lineage relationship between effector and memory CD4 T cells resembles that for CD8 T cells responding to the same pathogen. We find that, in parallel with effector CD8 T cells, IFN-gamma-positive effector CD4 T cells give rise to long-lived memory T cells capable of anamnestic responses to antigenic rechallenge.

GFP reporter mouse models of UPS proteolytic function

Kumarapeli and co-workers presented in the FASEB journal an in vivo reporter model for the ubiquitin/ proteasome system (UPS) (1). Similarly to the strategy that we used previously for the generation of our transgenic reporter model (2), they generated mice ubiquitously expressing a modified green fluorescent protein (GFP) that is constitutively targeted for proteasomal degradation. While we welcome new mouse models for the UPS, we regret that the authors, in an attempt to make a direct comparison of the two models based on the literature, misrepresented some data from our earlier study. The authors argue that the proteasome inhibitor provoked in their UPS reporter mice accumulation of the reporter in several tissues that did not respond in earlier experiments with our reporter mice (1). In addition, it is stated that this ‘unresponsiveness’ of tissues is a serious shortcoming of our reporter system. The authors correctly state that they used the same proteasome inhibitor (MG262), the same inhibitor concentration (5 mol/kg) and analyzed the mice at the same time after inhibitor administration (20 hrs), but fail to point out that they injected the inhibitor intravenously while we used intraperitoneal administration (1, 2). We feel it is important to point out to the readers that variations in responsiveness can be alternatively explained as a direct consequence of differences in the bioavailability of the inhibitor due to the route of administration. Moreover, we showed that several primary cultures obtained from our reporter mice (cardiomyocytes, neurons and fibroblasts) properly responded with GFP accumulation to treatment with proteasome inhibitors in vitro (2). Unfortunately, Kumarapeli and co-workers ignored this important observation even though they used the same approach to further validate the responsiveness in their own mouse model (1). We anticipate that, since these mouse models are based on different type of UPS substrates (3), it is indeed likely that there will be variations in the responsiveness of these model substrates (which may reflect how different classes of substrates are handled by the UPS) but would like to emphasize that the data presented by Kumarapeli and co-workers do not allow drawing conclusions on this important issue. We are pleased to see that the usage of GFP reporter substrates of the UPS is becoming more widespread. In vivo UPS models have been instrumental in gaining new insights in pathologic processes as well as actions of proteasome inhibitiors (3–5). We are convinced that, also in the future, the different UPS reporter mice will be important tools to decipher the role of the UPS in various pathologies and are looking forward to detailed studies with UPS reporter mice (6).