Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation

In spinal muscular atrophy (SMA), the leading genetic cause of early childhood death, the survival motor neuron 1 gene (SMN1) is deleted or inactivated. The nearly identical SMN2 gene has a silent mutation that impairs the utilization of exon 7 and the production of functional protein. It has been hypothesized that therapies boosting SMN2 exon 7 inclusion might prevent or cure SMA. Exon 7 inclusion can be stimulated in cell culture by oligonucleotides or intracellularly expressed RNAs, but evidence for an in vivo improvement of SMA symptoms is lacking. Here, we unambiguously confirm the above hypothesis by showing that a bifunctional U7 snRNA that stimulates exon 7 inclusion, when introduced by germline transgenesis, can efficiently complement the most severe mouse SMA model. These results are significant for the development of a somatic SMA therapy, but may also provide new means to study pathophysiological aspects of this devastating diseas

Release of human cytomegalovirus from latency by a KAP1/TRIM28 phosphorylation switch

Human cytomegalovirus (HCMV) is a highly prevalent pathogen that induces life-long infections notably through the establishment of latency in hematopoietic stem cells (HSC). Bouts of reactivation are normally controlled by the immune system, but can be fatal in immuno-compromised individuals such as organ transplant recipients. Here, we reveal that HCMV latency in human CD34(+) HSC reflects the recruitment on the viral genome of KAP1, a master co-repressor, together with HP1 and the SETDB1 histone methyltransferase, which results in transcriptional silencing. During lytic infection, KAP1 is still associated with the viral genome, but its heterochromatin-inducing activity is suppressed by mTOR-mediated phosphorylation. Correspondingly, HCMV can be forced out of latency by KAP1 knockdown or pharmacological induction of KAP1 phosphorylation, and this process can be potentiated by activating NFkB with TNF-α. These results suggest new approaches both to curtail CMV infection and to purge the virus from organ transplants

Chromosome Conformation Capture Uncovers Potential Genome-Wide Interactions between Human Conserved Non-Coding Sequences

Comparative analyses of various mammalian genomes have identified numerous conserved non-coding (CNC) DNA elements that display striking conservation among species, suggesting that they have maintained specific functions throughout evolution. CNC function remains poorly understood, although recent studies have identified a role in gene regulation. We hypothesized that the identification of genomic loci that interact physically with CNCs would provide information on their functions. We have used circular chromosome conformation capture (4C) to characterize interactions of 10 CNCs from human chromosome 21 in K562 cells. The data provide evidence that CNCs are capable of interacting with loci that are enriched for CNCs. The number of trans interactions varies among CNCs; some show interactions with many loci, while others interact with few. Some of the tested CNCs are capable of driving the expression of a reporter gene in the mouse embryo, and associate with the oligodendrocyte genes OLIG1 and OLIG2. Our results underscore the power of chromosome conformation capture for the identification of targets of functional DNA elements and raise the possibility that CNCs exert their functions by physical association with defined genomic regions enriched in CNCs. These CNC-CNC interactions may in part explain their stringent conservation as a group of regulatory sequences

Measuring In Vivo Protein Half-Life

Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Here, we demonstrate how chemical labeling of SNAP-tag fusion proteins can be exploited to measure the half-life of resident intracellular and extracellular proteins in living mice. First, we demonstrate that SNAP-tag substrates have wide bioavailability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then apply near-infrared probes to perform noninvasive imaging of in vivo-labeled tumors. Finally, we use SNAP-mediated chemical pulse-chase labeling to perform measurement of the in vivo half-life of different extra- and intracellular proteins. These results open broad perspectives for studying protein function in living animals

A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the olig1 and olig2 locus

Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers

Modulation of mitophagy and use thereof

The present invention relates to an agent or composition comprising at least one of: (i) a micro-RNA targeting a mitophagy-associated gene or a variant thereof, (ii) a vector comprising a nucleic acid encoding said micro-RNA or variant thereof, (iii) an inducer of said micro-RNA, and (iv) an inhibitor of said micro-RNA, for use as a medicament. In particular, the invention relates to substances and compositions useful in the treatment of mitochondria-related diseases, in a subject, or to enhance protocols of in vitro production of red blood cells

Vecteurs dérivés du virus de l' immunodéficience humaine de type 1 (VIH-1) pour le transfert de gène dans les cellules souches hématopoïétiques (mécanismes d' entrée et contrôle de l' expression)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Production and titration of lentiviral vectors

Lentiviral vectors have emerged over the last decade as powerful, reliable, and safe tools for stable gene transfer in a wide variety of mammalian cells. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells, including neurons. This is why lentivectors (LVs) are becoming the most useful and promising tools in the field of neuroscience, not only for research, but also for future gene and cell therapy approaches. LVs derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. These latest designs are reviewed in this unit. This unit also describes protocols for production and titration of LVs that can be implemented in a research laboratory setting, with an emphasis on standardization to improve transposability of results between laboratories

Lentiviral Vector Mediated Transgenesis

The genetic manipulation of rodents through the generation of fully transgenic animals or via the modification of selective cells or organs is a procedure of paramount importance for biomedical research, either to address fundamental questions or to develop preclinical models of human diseases. Lentiviral vectors occupy the front stage in this scene, as they can mediate the integration and stable expression of transgenes both in vitro and in vivo. Widely used to modify a variety of cells, including re-implantable somatic and embryonic stem cells, lentiviral vectors can also be directly administered in vivo, for instance in the brain. However, perhaps their most spectacular research application is in the generation of transgenic animals. Compared with the three-decade-old DNA pronuclear injection technique, lentivector-mediated transgenesis is simple, cheap, and highly efficient. Furthermore, it can take full advantage of the great diversity of lentiviral vectors developed for other applications, and thus allows for ubiquitous or tissue-specific or constitutive or externally controllable transgene expression, as well as RNAi-mediated gene knockdow