Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

BACKGROUND: Pharmacologic control of Cre-mediated recombination using tamoxifen-dependent activation of a Cre-estrogen receptor ligand binding domain fusion protein [CreER(T)] is widely used to modify and/or visualize cells in the mouse. METHODS AND FINDINGS: We describe here two new mouse lines, constructed by gene targeting to the Rosa26 locus to facilitate Cre-mediated cell modification. These lines should prove particularly useful in the context of sparse labeling experiments. The R26rtTACreER line provides ubiquitous expression of CreER under transcriptional control by the tetracycline reverse transactivator (rtTA); dual control by doxycycline and tamoxifen provides an extended dynamic range of Cre-mediated recombination activity. The R26IAP line provides high efficiency Cre-mediated activation of human placental alkaline phosphatase (hPLAP), complementing the widely used, but low efficiency, Z/AP line. By crossing with mouse lines that direct cell-type specific CreER expression, the R26IAP line has been used to produce atlases of labeled cholinergic and catecholaminergic neurons in the mouse brain. The R26IAP line has also been used to visualize the full morphologies of retinal dopaminergic amacrine cells, among the largest neurons in the mammalian retina. CONCLUSIONS: The two new mouse lines described here expand the repertoire of genetically engineered mice available for controlled in vivo recombination and cell labeling using the Cre-lox system

A Genetic Strategy for Stochastic Gene Activation with Regulated Sparseness (STARS)

It remains a challenge to establish a straightforward genetic approach for controlling the probability of gene activation or knockout at a desired level. Here, we developed a method termed STARS: stochastic gene activation with genetically regulated sparseness. The stochastic expression was achieved by two cross-linked, mutually-exclusive Cre-mediated recombinations. The stochastic level was further controlled by regulating Cre/lox reaction kinetics through varying the intrachromosomal distance between the lox sites mediating one of the recombinations. In mammalian cell lines stably transfected with a single copy of different STARS transgenes, the activation/knockout of reporter genes was specifically controlled to occur in from 5% to 50% of the cell population. STARS can potentially provide a convenient way for genetic labeling as well as gene expression/knockout in a population of cells with a desired sparseness level

Genetically-Directed, Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology

Background: In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes. Methods and Findings: In the present study we have addressed this application by using CreER technology to noninvasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT)-IRES-CreER or tyrosine hydroxylase (TH)-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL)-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2)-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species. Conclusions: Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful fo

Characterization of Parameters Required for Effective Use of Tamoxifen-Regulated Recombination

Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems. Ligand regulated forms of Cre have been increasingly used in these applications in order to gain temporal and spatial control over conditional targeting. The tamoxifen-regulated Cre variant mer-Cre-mer (mCrem) is widely utilized because of its reputation for tight regulation in the absence of its tamoxifen ligand. In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem. This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target. We find that robust mCrem expression correlates with a high level of tamoxifen-independent Cre activity, while clones expressing mCrem at the limit of western blot detection exhibit extremely tight regulation. We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population

Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice

Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreERTm and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1PB-CreERTm;R26RlacZ mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs

The Morphology and Intrinsic Excitability of Developing Mouse Retinal Ganglion Cells

The retinal ganglion cells (RGCs) have diverse morphology and physiology. Although some studies show that correlations between morphological properties and physiological properties exist in cat RGCs, these properties are much less distinct and their correlations are unknown in mouse RGCs. In this study, using three-dimensional digital neuron reconstruction, we systematically analyzed twelve morphological parameters of mouse RGCs as they developed in the first four postnatal weeks. The development of these parameters fell into three different patterns and suggested that contact from bipolar cells and eye opening might play important roles in RGC morphological development. Although there has been a general impression that the morphological parameters are not independent, such as RGCs with larger dendritic fields usually have longer but sparser dendrites, there was not systematic study and statistical analysis proving it. We used Pearson's correlation coefficients to determine the relationship among these morphological parameters and demonstrated that many morphological parameters showed high statistical correlation. In the same cells we also measured seven physiological parameters using whole-cell patch-clamp recording, focusing on intrinsic excitability. We previously reported the increase in intrinsic excitability in mouse RGCs during early postnatal development. Here we showed that strong correlations also existed among many physiological parameters that measure the intrinsic excitability. However, Pearson's correlation coefficient revealed very limited correlation across morphological and physiological parameters. In addition, principle component analysis failed to separate RGCs into clusters using combined morphological and physiological parameters. Therefore, despite strong correlations within the morphological parameters and within the physiological parameters, postnatal mouse RGCs had only limited correlation between morphology and physiology. This may be due to developmental immaturity, or to selection of parameters

Cre-Dependent Expression of Multiple Transgenes in Isolated Neurons of the Adult Forebrain

Background: Transgenic mice with mosaic, Golgi-staining-like expression of enhanced green fluorescent protein (EGFP) have been very useful in studying the dynamics of neuronal structure and function. In order to further investigate the molecular events regulating structural plasticity, it would be useful to express multiple proteins in the same sparse neurons, allowing co-expression of functional proteins or co-labeling of subcellular compartments with other fluorescent proteins. However, it has been difficult to obtain reproducible expression in the same subset of neurons for direct comparison of neurons expressing different functional proteins. Principal Findings: Here we describe a Cre-transgenic line that allows reproducible expression of transgenic proteins of choice in a small number of neurons of the adult cortex, hippocampus, striatum, olfactory bulb, subiculum, hypothalamus, superior colliculus and amygdala. We show that using these Cre-transgenic mice, multiple Cre-dependent transgenes can be expressed together in the same isolated neurons. We also describe a Cre-dependent transgenic line expressing a membrane associated EGFP (EGFP-F). Crossed with the Cre-transgenic line, EGFP-F expression starts in the adolescent forebrain, is present in dendrites, dendritic protrusions, axons and boutons and is strong enough for acute or chronic in vivo imaging. Significance: This triple transgenic approach will aid the morphological and functional characterization of neurons in various Cre-dependent transgenic mice

Tumor Spectrum, Tumor Latency and Tumor Incidence of the Pten-Deficient Mice

BACKGROUND: Pten functionally acts as a tumor suppressor gene. Lately, tissue-specific ablation of Pten gene in mice has elucidated the role of Pten in different tumor progression models. However, a temporally controlled Pten loss in all adult tissues to examine susceptibility of various tissues to Pten-deficient tumorigenesis has not been addressed yet. Our goal was to explore the genesis of Pten-deficient malignancies in multiple tissue lineages of the adult mouse. METHODS AND FINDINGS: We utilized an inducible Cre/loxP system to delete Pten exon 5 in the systemic organs of ROSA26 (R26)-CreER(T);Pten(fx/fx) mice. On reaching 45 weeks 4OHT-induced Pten loss, we found that the R26-CreER(T);Pten(fx/fx) mice developed a variety of malignancies. Overall tumor mean latency was 17 weeks in the Pten-deficient mice. Interestingly, mutant females developed malignancies more quickly at 10 approximately 11 weeks compared with a tumor latency of 21 weeks for mutant males. Lymphoma incidence (76.9% in females; 40.0% in males) was higher than the other malignancies found in the mutant mice. Mutant males developed prostate (20.0%), intestinal cancer (35.0%) and squamous cell carcinoma (10.0%), whereas the mutant females developed squamous cell carcinoma (15.4%) and endometrial cancer (46.1%) in addition to lymphomas. Furthermore, we tested the pharmacological inhibition of the PTEN downstream effectors using LY294002 on Pten-deficient prostate hyperplasia. Our data revealed that, indeed, the prostate hyperplasia resulting from the induced Pten loss was significantly suppressed by LY294002 (p = 0.007). CONCLUSIONS: Through monitoring a variety of Pten-deficient tumor formation, our results revealed that the lymphoid lineages and the epithelium of the prostate, endometrium, intestine and epidermis are highly susceptible to tumorigenesis after the Pten gene is excised. Therefore, this R26-CreER(T); Pten(fx/fx) mouse model may provide an entry point for understanding the role of Pten in the tumorigenesis of different organs and extend the search for potential therapeutic approaches to prevent Pten-deficient malignancies