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Antisense morpholino oligonucleotides have been commonly used in zebrafish to inhibit mRNA function, either by inhibiting pre-mRNA splicing or by blocking translation initiation. Even with the advent of genome editing by CRISP/Cas9 technology, morpholinos provide a useful and rapid tool to knockdown gene expression. This is especially true when dealing with multiple alleles and large gene families where genetic redundancy can complicate knockout of all family members. miRNAs are small noncoding RNAs that are often encoded in gene families and can display extensive genetic redundancy. This redundancy, plus their small size which can limit targeting by CRISPR/Cas9, makes morpholino-based strategies particularly attractive for inhibition of miRNA function. We provide the rationale, background, and methods to inhibit miRNA function with antisense morpholinos during early development and in the adult retina in zebrafish.
BACKGROUND - The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes.
RESULTS - We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9.
CONCLUSIONS - Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.
Damage of the zebrafish retina triggers a spontaneous regeneration response that is initiated by Müller Glia (MG) dedifferentiation and asymmetric cell division to produce multipotent progenitor cells. Subsequent expansion of the progenitor pool by proliferation is critical for retina regeneration. Pax6b expression in the progenitor cells is necessary for their proliferation, but exact regulation of its expression is unclear. Here, we show that miR-203 is downregulated during regeneration in proliferating progenitor cells. Elevated miR-203 levels inhibit progenitor cell expansion without affecting MG dedifferentiation or progenitor cell generation. Using GFP-reporter assays and gain and loss of function experiments in the retina, we show that miR-203 expression must be suppressed to allow pax6b expression and subsequent progenitor cell proliferation.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene-trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial- and temporal-specific gene inactivation but also permit spatial- and temporal-specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.
PURPOSE - Microneedles applied to the skin create micropores, allowing transdermal drug delivery of skin-impermeable compounds. The first human study with this technique demonstrated delivery of naltrexone (an opioid antagonist) for two to three days. Rapid micropore closure, however, blunts the delivery window. Application of diclofenac (an anti-inflammatory) allows seven days of naltrexone delivery in animals. The purpose of the current work was to demonstrate delivery of naltrexone for seven days following one microneedle treatment in humans.
METHODS - Human subjects were treated with microneedles, diclofenac (or placebo), and naltrexone. Impedance measurements were used as a surrogate marker to measure micropore formation, and plasma naltrexone concentrations were measured for seven days post-microneedle application.
RESULTS - Impedance dropped significantly from baseline to post-microneedle treatment, confirming micropore formation. Naltrexone was detected for seven days in Group 1 (diclofenac + naltrexone, n = 6), vs. 72 h in Group 2 (placebo + naltrexone, n = 2). At study completion, a significant difference in impedance was observed between intact and microneedle-treated skin in Group 1 (confirming the presence of micropores).
CONCLUSION - This is the first study demonstrating week-long drug delivery after one microneedle application, which would increase patient compliance and allow delivery of therapies for chronic diseases.
Drugs absorbed poorly through the skin are commonly delivered via injection with a hypodermic needle, which is painful and increases the risk of transmitting infectious diseases. Microneedles (MNs) selectively and painlessly permeabilize the outermost skin layer, allowing otherwise skin-impermeable drugs to cross the skin through micron-sized pores and reach therapeutic concentrations. However, rapid healing of the micropores prevents further drug delivery, blunting the clinical utility of this unique transdermal technique. We present the first human study demonstrating that micropore lifetime can be extended following MN treatment. Subjects received one-time MN treatment and daily topical application of diclofenac sodium. Micropore closure was measured with impedance spectroscopy, and area under the admittance-time curve (AUC) was calculated. AUC was significantly higher at MN+diclofenac sodium sites vs. placebo, suggesting slower rates of micropore healing. Colorimetry measurements confirmed the absence of local erythema and irritation. This mechanistic human proof-of-concept study demonstrates that micropore lifetime can be prolonged with simple topical administration of a non-specific cyclooxygenase inhibitor, suggesting the involvement of subclinical inflammation in micropore healing. These results will allow for longer patch wear time with MN-enhanced delivery, thus increasing patient compliance and expanding the transdermal field to a wider variety of clinical conditions.
Copyright © 2012 Elsevier B.V. All rights reserved.
The primate auditory cortex is comprised of a core region of three primary areas, surrounded by a belt region of secondary areas and a parabelt region lateral to the belt. The main sources of thalamocortical inputs to the auditory cortex are the medial geniculate complex (MGC), medial pulvinar (PM), and several adjoining nuclei in the posterior thalamus. The distribution of inputs varies topographically by cortical area and thalamic nucleus, but in a manner that has not been fully characterized in primates. In this study, the thalamocortical connections of the lateral belt and parabelt were determined by placing retrograde tracer injections into various areas of these regions in the marmoset monkey. Both regions received projections from the medial (MGm) and posterodorsal (MGpd) divisions of the medial geniculate complex (MGC); however, labeled cells in the anterodorsal (MGad) division were present only from injections into the caudal belt. Thalamic inputs to the lateral belt appeared to come mainly from the MGC, whereas the parabelt also received a strong projection from the PM, consistent with its position as a later stage of auditory cortical processing. The results of this study also indicate that the organization of the marmoset auditory cortex is similar to other primates.
Copyright © 2012 Wiley Periodicals, Inc.
The current working model of primate auditory cortex is constructed from a number of studies of both new and old world monkeys. It includes three levels of processing. A primary level, the core region, is surrounded both medially and laterally by a secondary belt region. A third level of processing, the parabelt region, is located lateral to the belt. The marmoset monkey (Callithrix jacchus jacchus) has become an important model system to study auditory processing, but its anatomical organization has not been fully established. In previous studies, we focused on the architecture and connections of the core and medial belt areas (de la Mothe et al., 2006a, J Comp Neurol 496:27-71; de la Mothe et al., 2006b, J Comp Neurol 496:72-96). In this study, the corticocortical connections of the lateral belt and parabelt were examined in the marmoset. Tracers were injected into both rostral and caudal portions of the lateral belt and parabelt. Both regions revealed topographic connections along the rostrocaudal axis, where caudal areas of injection had stronger connections with caudal areas, and rostral areas of injection with rostral areas. The lateral belt had strong connections with the core, belt, and parabelt, whereas the parabelt had strong connections with the belt but not the core. Label in the core from injections in the parabelt was significantly reduced or absent, consistent with the idea that the parabelt relies mainly on the belt for its cortical input. In addition, the present and previous studies indicate hierarchical principles of anatomical organization in the marmoset that are consistent with those observed in other primates.
Copyright © 2012 Wiley Periodicals, Inc.
KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.
Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level(1,2,3,) highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM)( 6). The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) (7, 8, 9,10,11). Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor(7,12). PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect (7, 10,11). The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.