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Find video protocols related to scientific articles indexed in Pubmed.
Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis.
FASEB J.
PUBLISHED: 11-05-2014
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Histone methylations have been implicated to play important roles in diverse cellular processes. Of particular interest is the methylation of histone H3K79, which is catalyzed by an evolutionarily conserved methyltransferase, disruptor of telomeric silencing (Dot1)-like (Dot1L). To investigate the role of Dot1L during vertebrate development, we have generated a Dot1L-specific transcription activator-like effector nuclease (TALEN) nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We show that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis because normally feeding tadpoles were formed, consistent with the lack of maternal Dot1L expression. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings suggest that Dot1L and H3K79 methylation play an important role for tadpole growth and development prior to metamorphosis into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of 2 separate phases of vertebrate development with distinct requirements for epigenetic modifications.-Wen, L., Fu, L., Guo, X., Chen, Y., Shi, Y.-B. Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis.
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RAG1/2 knockout pigs with severe combined immunodeficiency.
J. Immunol.
PUBLISHED: 06-27-2014
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Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.
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Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique.
Sci Rep
PUBLISHED: 03-26-2014
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Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 ? subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.
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The asymmetric influence of the positive and negative IOD events on China's rainfall.
Sci Rep
PUBLISHED: 02-05-2014
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Rainfall anomalies over southern China are found to be asymmetricly influenced by the Indian Ocean Dipole (IOD), with a far stronger influence from positive IOD (pIOD) events. A greater convection anomaly and an equivalent-barotropic Rossby wave train response occurs during pIOD events than during negative IOD (nIOD) events. Over the Bay of Bengal (BOB) and South China Sea (SCS), an associated low-level anomalous anticyclone strengthens the southwesterlies during boreal fall (September, October and November, SON), when a pIOD matures. The increased moisture flux gives rise to the anomalously high rainfall over southern China. During its developing phase (boreal summer, June, July, and August, JJA), the influence of a pIOD event on the contemporaneous rainfall over southern China is weak, but a JJA pIOD index is highly correlated with fall rainfall. Therefore, this index can serve as a potential predictor for variations of boreal fall rainfall over southern China.
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Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.
Development
PUBLISHED: 01-08-2014
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For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.
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Generation of knockout rabbits using transcription activator-like effector nucleases.
Cell Regen (Lond)
PUBLISHED: 01-01-2014
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Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.
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Cypher/ZASP is a novel A-kinase anchoring protein.
J. Biol. Chem.
PUBLISHED: 08-31-2013
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PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RII? of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser(265) and Ser(296) by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser(1928) induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.
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Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos.
Cell Biosci
PUBLISHED: 01-23-2013
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Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.
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Left radial access is preferable to right radial access for the diagnostic or interventional coronary procedures: a meta-analysis involving 22 randomized clinical trials and 10287 patients.
PLoS ONE
PUBLISHED: 01-01-2013
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The transradial approach has been used extensively for both diagnostic and interventional coronary procedures; however, there is no universal consensus hitherto on the optimal choice of radial access from either the left or the right artery. We therefore sought to meta-analyze available randomized clinical trials to compare the left with the right radial access for the diagnostic or interventional coronary procedures.
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Retinoic acid-activated Ndrg1a represses Wnt/?-catenin signaling to allow Xenopus pancreas, oesophagus, stomach, and duodenum specification.
PLoS ONE
PUBLISHED: 01-01-2013
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How cells integrate multiple patterning signals to achieve early endoderm regionalization remains largely unknown. Between gastrulation and neurulation, retinoic acid (RA) signaling is required, while Wnt/?-catenin signaling has to be repressed for the specification of the pancreas, oesophagus, stomach, and duodenum primordia in Xenopus embryos. In attempt to screen for RA regulated genes in Xenopus endoderm, we identified a direct RA target gene, N-myc downstream regulated gene 1a (ndrg1a) that showed expression early in the archenteron roof endoderm and late in the developing pancreas, oesophagus, stomach, and duodenum. Both antisense morpholino oligonucleotide mediated knockdown of ndrg1a in Xenopus laevis and the transcription activator-like effector nucleases (TALEN) mediated disruption of ndrg1 in Xenopus tropicalis demonstrate that like RA signaling, Ndrg1a is specifically required for the specification of Xenopus pancreas, oesophagus, stomach, and duodenum primordia. Immunofluorescence data suggest that RA-activated Ndrg1a suppresses Wnt/?-catenin signaling in Xenopus archenteron roof endoderm cells. Blocking Wnt/?-catenin signaling rescued Ndrg1a knockdown phenotype. Furthermore, overexpression of the putative Wnt/?-catenin target gene Atf3 phenocopied knockdown of Ndrg1a or inhibition of RA signaling, while Atf3 knockdown can rescue Ndrg1a knockdown phenotype. Lastly, the pancreas/stomach/duodenum transcription factor Pdx1 was able to rescue Atf3 overexpression or Ndrg1a knockdown phenotype. Together, we conclude that RA activated Ndrg1a represses Wnt/?-catenin signaling to allow the specification of pancreas, oesophagus, stomach, and duodenum progenitor cells in Xenopus embryos.
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Developmental expression of sideroflexin family genes in Xenopus embryos.
Dev. Dyn.
PUBLISHED: 08-26-2010
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Emerging evidence indicates that mitochondrial carriers are not only crucial for metabolism, but also important for embryonic development. Sideroflexin is a novel family of mitochondrial tricarboxylate carrier proteins, of which the in vivo function is largely unknown. Here, we report on the expression patterns of five sideroflexin genes in Xenopus embryos. Whole-mount in situ hybridization analysis reveals that while sideroflexin2 is expressed in the pancreas, sideroflexin1 and 3 display a complex expression in the central nervous system, somites, pronephros, liver, and pancreas. In contrast, only a weak expression of sideroflexin4 and 5 was detected in embryonic brain. Taken together, the five sideroflexin genes show both overlapping and nonoverlapping expression during Xenopus embryogenesis. As the primary structures of the five sideroflexin proteins are also quite similar, their functional redundancy should be taken into consideration for gene targeting studies.
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Low molecular weight heparin inhibits circulating fibrocytes differentiation by modulating neuronal nitric oxide synthase and TGF-?1/Smad pathway.
Cell. Physiol. Biochem.
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Circulating fibrocytes (CFs) have been placed at the center of a number of fibrosing conditions. Recently, attention has been drawn to the non-anticoagulant activities of low molecular weight heparin (LH), especially its anti-fibrotic effects. The purpose of this study was to investigate the effects of LH on CFs differentiation and possible underlying mechanisms.
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Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).
Proc. Natl. Acad. Sci. U.S.A.
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Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.
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IKs protects from ventricular arrhythmia during cardiac ischemia and reperfusion in rabbits by preserving the repolarization reserve.
PLoS ONE
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The function of the repolarization reserve in the prevention of ventricular arrhythmias during cardiac ischemia/reperfusion and the impact of ischemia on slowly activated delayed rectifier potassium current (I(Ks)) channel subunit expression are not well understood.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.