24 research outputs found
Synthesis of 15-RVD GoldyTALENs.
<p>A schematic diagram showing the assembly of 15-RVD GoldyTALENs by the Golden Gate method described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Cermak1" target="_blank">[17]</a>. The RCIscript-GoldyTALEN backbone, all other component plasmids (as Golden Gate TALEN kit 2.0), and the 256 pFUS_B4 clones are distributed through Addgene. *XX denoted either NI, HD, NN or NG.</p
Somatic efficacy of 15-RVD TALEN compared with other reported TALEN scaffolds, ZFN and CRISPR/Cas9.
<p>Plot of somatic mutation rates in zebrafish genome editing using ZFNs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Sander1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Chen2" target="_blank">[32]</a>, TALENs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Cade1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Moore1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Sander2" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Chen2" target="_blank">[32]</a> and CRISPR/Cas9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065259#pone.0065259-Hwang1" target="_blank">[46]</a> from representative publications together with 15-RVD TALENs from this study. *Only Homo-dimeric TALENs were compared. <sup>†</sup>Only 15-RVD TALENs with spacer lengths between 13–20 bp and a T nucleotide preceding the TALEN binding site were included.</p
Importance of the spacer length and the 5′ T nucleotide in GoldyTALEN activity.
<p>(A) Modifications of NPMB1 P1 to LS and RS for longer spacers. (B) Both modified NPMBP1 TALEN pairs (NPM1B P1 LS and RS) showed significant increase in <i>in vivo</i> activity compared with the original shorter spacer design. (C) Modifications of IDH1 P1 and JAK2A P1 to remove the 5′ upstream T nucleotide at one of the TALEN arms. (D) Both modified TALEN pairs (IDH1 P1 RM and JAK2A P1 LM) showed significant reduction in <i>in vivo</i> activity compared with the original designs. Open arrowheads indicate bands from completely digested WT PCR product and closed arrowheads represent uncut PCR product with small indels. WT: wild-type; T: TALEN pair injected. Representative and average results of RFLP screening in 3 separate experiments analyzing group of 10 embryos are shown in the gel photo and graph, respectively. Error bars represent the standard error of the means.</p
Deletion of large genomic fragments with two pairs of TALEN.
<p>(A) Schematic diagram showing the strategy of introducing large genomic deletions at <i>flt3</i> and <i>jak2a</i> loci using two TALEN pairs. Green and purple arrows represent primer pairs used for PCR screening of corresponding large deletion. WT: wild-type; T: TALEN pairs injected. (B) Sequences of germline-transmitted large deletions in F1 embryos. Sequence of a single mutant F1 embryo from each founder is shown. Underlined are TALEN binding sites. Sequences in blue represent insertions.</p
Design of 15-RVD GoldyTALENs.
<p>The design parameters of active 15-RVD GoldyTALEN pairs used in this study. Each TALEN arm consists of a DNA binding domain with 15-RVDs (14.5 TALE repeats), corresponding to 15-nucleotides DNA binding sequence proceeded by a 5′ T nucleotides and a 13 to 20 bp spacer in between 2 arms containing a restriction recognition sequence to assay activity. *Initial design parameter was spacer length between 11–20 bp and only the two inactive pairs, NPM1B P1 and P2 have spacers shorter than 13 bp.</p
Correlation of spacer lengths and somatic activities of 15-RVD GoldyTALENs.
*<p>shown in decreasing order of activity.</p>†<p>modified without the 5′ T nucleotide at one of the TALEN arms.</p
An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish
<div><h3>Background</h3><p>Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.</p> <h3>Methods and Results</h3><p>Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of <em>vegfc</em> and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.</p> <h3>Conclusion</h3><p>Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.</p> </div
Quantification of lymphatic capillary development after stimulation with rhVEGF-C.
<p>Red fluorescent images from late-phase microangiograms of <i>Tg(fli1:EGFP)<sup>y1</sup></i> zebrafish are shown, highlighting Texas Red-LMD uptake within lymphatic capillaries. Results of quantitative morphometric analyses are displayed in bar graphs. <b>A</b> and <b>B</b>, At 2 dpf, fewer lymphatic capillaries are present in untreated zebrafish (n = 18), compared to 3-dpf untreated zebrafish (n = 32), but they increase significantly with the addition of rhVEGF-C to the zebrafish aqueous environment (n = 18). *P<0.0001, **P<0.0001. Scale bars, 50 µm.</p
Temporal expression patterns of eCB receptor genes.
<p>The time points on all graphs are represented as means ± 95% CI (0.25–1 dpf: 20 larvae/n, n = 3; 2–7 dpf: 10 larvae/n, n = 3). * Indicates that a group is significantly different from the 5 dpf group (Sidak's multiple comparisons test, <i>P <</i> 0.05). (A) The fold change of <i>cnr1</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (B) The fold change of <i>cnr2</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (C) The fold change of <i>loc793909</i> transcript levels relative to 5 dpf as determined by qRT-PCR.</p
The endocannabinoid gene <i>faah2a</i> modulates stress-associated behavior in zebrafish
<div><p>The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (<i>abhd4</i>, <i>gde1</i>, <i>napepld)</i>, enzymes that degrade (<i>faah</i>, <i>faah2a</i>, <i>faah2b</i>), and receptors that bind (<i>cnr1</i>, <i>cnr2</i>, <i>gpr55-like</i>) endocannabinoids are expressed in zebrafish (<i>Danio rerio</i>). These genes are conserved in many other vertebrates, including humans, but fatty acid amide hydrolase 2 has been lost in mice and rats. We engineered transcription activator-like effector nucleases to create zebrafish with mutations in <i>cnr1</i> and <i>faah2a</i> to test the role of these genes in modulating stress-associated behavior. We showed that disruption of <i>cnr1</i> potentiated locomotor responses to hyperosmotic stress. The increased response to stress was consistent with rodent literature and served to validate the use of zebrafish in this field. Moreover, we showed for the first time that disruption of <i>faah2a</i> attenuated the locomotor responses to hyperosmotic stress. This later finding suggests that FAAH2 may be an important mediator of stress responses in non-rodent vertebrates. Accordingly, FAAH and FAAH2 modulators could provide distinct therapeutic options for stress-aggravated disorders.</p></div