Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting

A cre-inducible DUX4 transgenic mouse model for investigating facioscapulohumeral muscular dystrophy

The Double homeobox 4 (DUX4) gene is an important regulator of early human development and its aberrant expression is causal for facioscapulohumeral muscular dystrophy (FSHD). The DUX4-full length (DUX4-fl) mRNA splice isoform encodes a transcriptional activator; however, DUX4 and its unique DNA binding preferences are specific to old-world primates. Regardless, the somatic cytotoxicity caused by DUX4 expression is conserved when expressed in cells and animals ranging from fly to mouse. Thus, viable animal models based on DUX4-fl expression have been difficult to generate due in large part to overt developmental toxicity of low DUX4-fl expression from leaky transgenes. We have overcome this obstacle and here we report the generation and initial characterization of a line of conditional floxed DUX4-fl transgenic mice, FLExDUX4, that is viable and fertile. In the absence of cre, these mice express a very low level of DUX4-fl mRNA from the transgene, resulting in mild phenotypes. However, when crossed with appropriate cre-driver lines of mice, the double transgenic offspring readily express DUX4-fl mRNA, protein, and target genes with the spatiotemporal pattern of nuclear cre expression dictated by the chosen system. When cre is expressed from the ACTA1 skeletal muscle-specific promoter, the double transgenic animals exhibit a developmental myopathy. When crossed with tamoxifen-inducible cre lines, DUX4-mediated pathology can be induced in adult animals. Thus, the appearance and progression of pathology can be controlled to provide readily screenable phenotypes useful for assessing therapeutic approaches targeting DUX4-fl mRNA and protein. Overall, the FLExDUX4 line of mice is quite versatile and will allow new investigations into mechanisms of DUX4-mediated pathophysiology as well as much-needed pre-clinical testing of DUX4-targeted FSHD interventions in vivo

The fatty acid binding protein 7 (FABP7) is involved in proliferation and invasion of melanoma cells

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms underlying melanoma tumor development and progression are still not completely understood. One of the new candidates that emerged from a recent gene expression profiling study is <it>fatty acid-binding protein 7 </it>(<it>FABP7)</it>, involved in lipid metabolism, gene regulation, cell growth and differentiation.</p> <p>Methods</p> <p>We studied the functional role of FABP7 in human melanoma cell lines and using immunohistochemistry analyzed its expression pattern and clinical role in 11 nevi, 149 primary melanomas and 68 metastases.</p> <p>Results</p> <p>FABP7 mRNA and protein level is down-regulated following treatment of melanoma cell lines with a PKC activator (PMA) or MEK1 inhibitor (PD98059). Down-regulation of FABP7 using siRNA decreased cell proliferation and invasion but did not affect apoptosis. In clinical specimens, FABP7 was expressed in 91% of nevi, 71% of primary melanomas and 70% of metastases, with a cytoplasmic and/or nuclear localization. FABP7 expression was associated with tumor thickness in superficial spreading melanoma (P = 0.021). In addition, we observed a trend for an association between FABP7 expression and Ki-67 score (P = 0.070) and shorter relapse-free survival (P = 0.069) in this group of patients.</p> <p>Conclusion</p> <p>Our data suggest that FABP7 can be regulated by PKC and the MAPK/ERK1/2 pathway through independent mechanisms in melanoma cell lines. Furthermore, FABP7 is involved in cell proliferation and invasion <it>in vitro</it>, and may be associated with tumor progression in melanoma.</p

The REST remodeling complex protects genomic integrity during embryonic neurogenesis

The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation

Modeling Insertional Mutagenesis Using Gene Length and Expression in Murine Embryonic Stem Cells

Background. High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs) is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of genetrapping events have not been tested on a genome-wide scale. Methodology/Principal Findings. In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identifie

Postnatal Changes in the Expression Pattern of the Imprinted Signalling Protein XLαs Underlie the Changing Phenotype of Deficient Mice

The alternatively spliced trimeric G-protein subunit XLαs, which is involved in cAMP signalling, is encoded by the Gnasxl transcript of the imprinted Gnas locus. XLαs deficient mice show neonatal feeding problems, leanness, inertia and a high mortality rate. Mutants that survive to weaning age develop into healthy and fertile adults, which remain lean despite elevated food intake. The adult metabolic phenotype can be attributed to increased energy expenditure, which appears to be caused by elevated sympathetic nervous system activity. To better understand the changing phenotype of Gnasxl deficient mice, we compared XLαs expression in neonatal versus adult tissues, analysed its co-localisation with neural markers and characterised changes in the nutrient-sensing mTOR1-S6K pathway in the hypothalamus. Using a newly generated conditional Gnasxl lacZ gene trap line and immunohistochemistry we identified various types of muscle, including smooth muscle cells of blood vessels, as the major peripheral sites of expression in neonates. Expression in all muscle tissues was silenced in adults. While Gnasxl expression in the central nervous system was also developmentally silenced in some midbrain nuclei, it was upregulated in the preoptic area, the medial amygdala, several hypothalamic nuclei (e.g. arcuate, dorsomedial, lateral and paraventricular nuclei) and the nucleus of the solitary tract. Furthermore, expression was detected in the ventral medulla as well as in motoneurons and a subset of sympathetic preganglionic neurons of the spinal cord. In the arcuate nucleus of Gnasxl-deficient mice we found reduced activity of the nutrient sensing mTOR1-S6K signalling pathway, which concurs with their metabolic status. The expression in these brain regions and the hypermetabolic phenotype of adult Gnasxl-deficient mice imply an inhibitory function of XLαs in energy expenditure and sympathetic outflow. By contrast, the neonatal phenotype of mutant mice appears to be due to a transient role of XLαs in muscle tissues

Use of a conditional Ubr5 mutant allele to investigate the role an N-end rule ubiquitin-protein ligase in Hedgehog signalling and embryonic limb development

Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling