Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific ...regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx
mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.
Department of Fisheries, Animal and Veterinary Science, University of Rhode Island, Kingston, Rhode Island
Submitted 16 December 2008
; accepted in final form 18 May 2009
Deletion or inhibition of ...myostatin in mammals has been demonstrated to markedly increase muscle mass by hyperplasia, hypertrophy, or a combination of both. Despite a remarkably high degree of conservation with the mammalian protein, the function of myostatin remains unknown in fish, many species of which continue muscle growth throughout the lifecycle by hyperplasia. Transgenic rainbow trout ( Oncorhynchus mykiss ) overexpressing follistatin, one of the more efficacious antagonists of myostatin, were produced to investigate the effect of this protein on muscle development and growth. P 1 transgenics overexpressing follistatin in muscle tissue exhibited increased epaxial and hypaxial muscling similar to that observed in double-muscled cattle and myostatin null mice. The hypaxial muscling generated a phenotype reminiscent of well-developed rectus abdominus and intercostal muscles in humans and was dubbed "six pack." Body conformation of the transgenic animals was markedly altered, as measured by condition factor, and total muscle surface area increased. The increased muscling was due almost exclusively to hyperplasia as evidenced by a higher number of fibers per unit area and increases in the percentage of smaller fibers and the number of total fibers. In several individuals, asymmetrical muscling was observed, but no changes in mobility or behavior of follistatin fish were observed. The findings indicate that overexpression of follistatin in trout, a species with indeterminate growth rate, enhances muscle growth. It remains to be determined whether the double muscling in trout is due to inhibition of myostatin, other growth factors, or both.
myostatin; growth; transgenic; Oncorhynchus mykiss
Address for reprint requests and other correspondence: T. M. Bradley, Dept. of Fisheries, Animal and Veterinary Science, Univ. of Rhode Island, East Farm, Bldg. #14, Route 108, Kingston, RI 02881 (e-mail: tbradley{at}uri.edu )
Aberrant activation of the receptor tyrosine kinase-mediated RAS signaling cascade is the primary driver of embryonal rhabdomyosarcoma (ERMS), a pediatric cancer characterized by a block in myogenic ...differentiation. To investigate the cellular function of activated RAS signaling in regulating the growth and differentiation of ERMS cells, we genetically ablated activated RAS oncogenes with high-efficiency genome-editing technology. Knockout of NRAS in CRISPR-inducible ERMS xenograft models resulted in near-complete tumor regression through a combination of cell death and myogenic differentiation. Utilizing this strategy for therapeutic RAS targeting in ERMS, we developed a recombinant oncolytic myxoma virus (MYXV) engineered with CRISPR/Cas9 gene-editing capability. Treatment of pre-clinical human ERMS tumor xenografts with an NRAS-targeting version of this MYXV significantly reduced tumor growth and increased overall survival. Our data suggest that targeted gene-editing cancer therapies have promising translational applications, especially with improvements to gene-targeting specificity and oncolytic vector technology.
Neurobiology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033
Submitted 17 November 2003
; accepted in final form 5 April 2004
Hypotonic stimulation induces airway constriction in ...normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca 2+ influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4 -phorbol 12,13-didecanoate (4- PDD), a TRPV4 ligand, increased intracellular Ca 2+ level only when Ca 2+ was present in the extracellular solution. The 4- PDD-induced Ca 2+ response was inhibited by ruthenium red (1 µM), a known TRPV4 inhibitor, but not by capsazepine (1 µM), a TRPV1 antagonist, indicating that 4- PDD-induced Ca 2+ response is mediated by TRPV4. Verapamil (10 µM), an L-type voltage-gated Ca 2+ channel inhibitor, had no effect on the 4- PDD-induced Ca 2+ response, excluding the involvement of L-type Ca 2+ channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca 2+ channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca 2+ channel inhibitor nifedipine (1 µM) or by the TRPV1 inhibitor capsazepine (1 µM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.
calcium influx; fluorometric imaging plate reader; hypotonic solution; smooth muscle contraction; vanilloid receptor-related osmotically activated channel
Address for reprint requests and other correspondence: Y. Jia, Neurobiology, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033 (E-mail: yanlin.jia{at}spcorp.com )
Context-Dependent Recognition Memory Murnane, Kevin; Phelps, Matthew P; Malmberg, Kenneth
Journal of experimental psychology. General,
12/1999, Letnik:
128, Številka:
4
Journal Article
Recenzirano
A solution to the problem of context-dependent recognition
memory is presented in terms of the item, associated context, and
ensemble (ICE) theory. It is argued that different types of context
...effects depend on how context information is encoded at both
learning and retrieval. Matching associated context in memory and a
retrieval cue produces increases in both hit and false alarm rates
and may not be accompanied by a change in discrimination.
Integrating item and context information in an ensemble and matching
ensemble information in memory and a retrieval cue produces
context-dependent discrimination. Empirical support for these
predictions is presented.
Background:
Test frequency, stakes associated with educational tests, and feedback from test results have been identified in the research literature as relevant factors in student achievement.
...Objectives:
Summarize the separate and joint contribution to student achievement of these three treatments and their interactions via multivariable meta-analytic techniques using a database of English-language studies spanning a century (1910–2010), comprising 149 studies and 509 effect size estimates.
Research design:
Analysis employed robust variance estimation. Considered as potential moderators were hundreds of study features comprising various test designs and test administration, demographic, and source document characteristics.
Subjects:
Subjects were students at all levels, from early childhood to adult, mostly from the United States but also eight other countries.
Results:
We find a summary effect size of 0.84 for the three treatments collectively. Further analysis suggests benefits accrue to the incremental addition of combinations of testing and feedback or stakes and feedback. Moderator analysis shows higher effect sizes associated with the following study characteristics: more recent year of publication, summative (rather than formative) testing, constructed (rather than selected) item response formats, alignment of subject matter between pre- and posttests, and recognition/recall (rather than core subjects, art, or physical education). Conversely, lower effect sizes are associated with postsecondary students (rather than early childhood–upper secondary), special education population, larger study population, random assignment (rather than another sampling method), use of shadow test as outcome measure, designation of individuals (rather than groups) as units of analysis, and academic (rather than corporate or government) research.
As the conservation challenges increase, new approaches are needed to help combat losses in biodiversity and slow or reverse the decline of threatened species. Genome‐editing technology is changing ...the face of modern biology, facilitating applications that were unimaginable only a decade ago. The technology has the potential to make significant contributions to the fields of evolutionary biology, ecology, and conservation, yet the fear of unintended consequences from designer ecosystems containing engineered organisms has stifled innovation. To overcome this gap in the understanding of what genome editing is and what its capabilities are, more research is needed to translate genome‐editing discoveries into tools for ecological research. Emerging and future genome‐editing technologies include new clustered regularly interspaced short palindromic repeats (CRISPR) targeted sequencing and nucleic acid detection approaches as well as species genetic barcoding and somatic genome‐editing technologies. These genome‐editing tools have the potential to transform the environmental sciences by providing new noninvasive methods for monitoring threatened species or for enhancing critical adaptive traits. A pioneering effort by the conservation community is required to apply these technologies to real‐world conservation problems.
Transformación de la Ecología y la Biología de la Conservación por medio de la Edición Genómica
Resumen
Conforme aumentan los retos de conservación, se necesitan nuevas estrategias para ayudar a combatir las pérdidas de biodiversidad y para disminuir o revertir la declinación de especies. La tecnología de edición genómica está cambiando el rostro de la biología moderna, facilitando aplicaciones que eran inimaginables hace una década. Esta tecnología tiene el potencial de contribuir significativamente en los campos de la biología evolutiva, la ecología y la conservación, aun así, el miedo a las consecuencias accidentales de los ecosistemas planeados que contienen organismos diseñados ha sofocado a la innovación. Para sobreponerse a este vacío en el entendimiento de lo que es la edición genómica y cuáles son sus capacidades se requiere de mayor investigación para traducir los descubrimientos de la edición genómica a herramientas para la investigación ecológica. Las tecnologías de edición genómica emergentes y futuras incluyen nuevas estrategias CRISPR enfocadas en la secuenciación y detección de ácidos nucleicos, así como tecnologías de definición del código de barras genético de las especies y de edición somática de genes. Estas herramientas de edición genómica tienen el potencial para transformar las ciencias ambientales al proporcionar nuevos métodos no invasivos para el monitoreo de especies amenazadas o para mejorar las características adaptativas más importantes. Se requiere de un esfuerzo vanguardista por parte de la comunidad conservadora para aplicar esta tecnología a los problemas de conservación en el mundo real.
摘要
随着生物多样性保护面临越来越多的挑战, 我们需要新的方法来对抗生物多样性丧失、减缓或逆转受胁迫物种的种群下降。基因组编辑技术推动了十年前人们还无法想象的应用, 正在改变着现代生物学的面貌。这项技术能为进化生物学、生态学和保护生物学领域做出重大贡献。然而, 对含有被工程改造过的生物有机体的生态系统是否会产生意外后果的担忧, 限制了这个方向的创新。为了帮助理解基因组编辑的概念及其功能, 还需要更多的研究将基因组编辑发现转变为生态学研究的工具。新兴及未来的基因组编辑技术包括新的 CRISPR 靶向测序和核酸检测方法、物种遗传条形码和体细胞基因组编辑技术等。这些基因组编辑工具可以通过提供新的非损伤性方法来监测受胁迫物种, 或改进关键的适应性性状, 为环境科学带来重大改变。因此, 要将这些技术应用于现实中的保护问题, 需要保护领域努力进行开拓。【翻译: 胡怡思; 审校: 聂永刚】
Article impact statement: Novel applications of CRISPR technology have the potential to make significant contributions to ecology and conservation.
Live foods used in a marine fish hatchery must provide the nutrients needed for the rapidly developing larvae. Marine pelagic fish eggs can contain up to 50% of their total amino acid pool as free ...amino acids (FAA), of which most is used up shortly after the onset of exogenous feeding as a source of energy. Copepod nauplii are richer in FAA than many traditional foods given to marine fish larvae. However, copepod nauplii can be difficult to produce in mass quantities in the hatchery and are more readily available by capture from the wild. The process of capture and acclimation may impact the nutrient quality of such nauplii. This study describes the FAA profile of nauplii of
Apocyclops panamensis collected from low salinity fertilized brackish-water ponds, the effects of acclimation from 6.6‰ to 30‰ seawater on the FAA profile, and the feasibility of direct enrichment of nauplii with isoleucine, leucine, lysine and valine. Nauplii were exposed to four treatment protocols over a 3
h period and sampled for FAA profiles, 1) no salinity change, 2) direct transfer to 30‰, 3) acclimated to 30‰, and 4) acclimated
+
FAA enrichment. Nauplii collected from 6.6‰ salinity ponds had a mean total FAA content of 27.22
±
5.85
nmol
·
mg
−1 w.w. nauplii at the time of collection. Forty-two percent of the total was comprised of essential amino acids of which arginine, lysine, leucine and valine were the most prevalent, and 55% were non-essential amino acids, primarily glutamic acid, alanine, proline, asparagine and glycine. A direct transfer of nauplii from a salinity of 6.6‰ to 30‰ resulted in a significant reduction in total FAA (TFAA) particularly in essential FAA (EAA). Acclimation of nauplii from a salinity of 6.6‰ to 30‰ over 3
h resulted in a significant increase in TFAA and particularly in non-essential FAA (NEAA). The addition of isoleucine, leucine, lysine and valine to the holding water during acclimation further increased their concentrations in the nauplii after a 3
h exposure.
Genetic and epigenetic changes in components of the Reelin-signaling pathway (RELN, DAB1) are associated with autism spectrum disorder (ASD) risk. Social communication deficits are a key component of ...the ASD diagnostic criteria, but the underlying neurogenetic mechanisms remain unknown. Reln insufficient mice exhibit ASD-like behavioral phenotypes including altered neonatal vocalization patterns. Reelin affects multiple pathways including through the receptors, Very low-density lipoprotein receptor (Vldlr), Apolipoprotein receptor 2 (Apoer2), and intracellular signaling molecule Disabled-1 (Dab1). As Vldlr was previously implicated in avian vocalization, here we investigate vocalizations of neonatal mice with a reduction or absence of these components of the Reelin-signaling pathway. Mice with low or no Dab1 expression exhibited reduced calling rates, altered call-type usage, and differential vocal development trajectories. Mice lacking Vldlr expression also had altered call repertoires, and this effect was exacerbated by deficiency in Apoer2. Together with previous findings, these observations 1) solidify a role for Reelin in vocal communication of multiple species, 2) point to the canonical Reelin-signaling pathway as critical for development of normal neonatal calling patterns in mice, and 3) suggest that mutants in this pathway could be used as murine models for Reelin-associated vocal deficits in humans.
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