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A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray
摘要： A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.
关键词： gene expression,enzyme-free,fluorescence microscopy,mRNA detection,microarray
更新于2025-11-21 11:24:58
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Mapping Neurotransmitter Identity in the Whole-Mount <i>Drosophila</i> Brain Using Multiplex High-Throughput Fluorescence <i>in Situ</i> Hybridization
摘要： Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence in situ hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount Drosophila melanogaster brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in Drosophila brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.
关键词： Neurotransmitter,mRNA,Fluorescence in situ hybridization,Gene expression,Drosophila
更新于2025-11-21 11:08:12
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Near-infrared upconversion–activated CRISPR-Cas9 system: A remote-controlled gene editing platform
摘要： As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light–responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as “nanotransducers” that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light–activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.
关键词： cancer therapeutics,upconversion nanoparticles,gene editing,CRISPR-Cas9,near-infrared
更新于2025-11-21 11:08:12
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Light Controlled In Vitro Gene Delivery Using Polymer-Tethered Spiropyran as a Photoswitchable Photosensitizer
摘要： A gene delivery system using spiropyran as a photoswitchable photosensitizer for controlled photochemical internalization effect was developed by engineering the outer coating of polyethylenimine/DNA complex with a small amount of spiropyran-containing cationic copolymers. The successful binding of cationic polymers by the polyethylenimine coating was detected by the distance-sensitive fluorescence resonance energy transfer technique that evidenced occurrence of energy transfer between fluorescein-labeled cationic copolymers and polyethylenimine-condensed rhodamine-labeled DNA. The ternary polyplexes feature reversible controllability of singlet oxygen generation based on the dual effect of spiropyrans in photochromism and aggregation-induced enhanced photosensitization, allowing significant light-induced amplification of bPEI-mediated in vitro transgene efficiency (from original 15% to final 91%) at a low DNA dose, with the integrity of supercoiled DNA structure unaffected. The use of spiropyran without the need of other photosensitizers circumvents the issue of uncontrolled long-lasting photocytotoxicity in gene delivery.
关键词： gene delivery,spiropyran,photoswitching,singlet oxygen,fluorescence resonance energy transfer
更新于2025-11-21 11:08:12
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In Vivo Fluorescence Retinal Imaging Following AAV2-Mediated Gene Delivery in the Rat Retina
摘要： PURPOSE. The purpose of this study was to evaluate longitudinal gene expression patterns by in vivo retinal imaging using a modified custom-built confocal laser-scanning microscope in experimental rats after intravitreal injection of recombinant adeno-associated virus 2 (rAAV2-green fluorescent protein [GFP]). METHODS. Ten 9-week-old Wistar rats were divided into two groups: experimental group (group 1) that received a rAAV2-GFP intravitreal injection and control group (group 2) that received a vehicle. After anesthesia using a Zoletil intraperitoneal injection, 8 μL rAAV2-GFP in group 1 or vehicle in group 2 was injected intravitreally using a 33-G Hamilton syringe. In vivo fluorescence retinal images were acquired under anesthesia at 2, 4, 6, and 13 days after rAAV or vehicle delivery. RESULTS. Differences in GFP fluorescence were identified starting from day 2 after the intravitreal injection of rAAV2-GFP in group 1. Between days 4 and 6, the intensity and area of fluorescence in the retina began to increase and peaked at day 13. Based on the pattern of GFP expression, the axon of the nerve fiber layer ganglion cell was identified. In group 2, eyes treated with the vehicle showed a small amount of autofluorescence in a limited area for up to 2 weeks, with no increase in intensity during this period. CONCLUSIONS. In vivo retinal imaging confirmed gene expression within 2 weeks after the intravitreal injection of rAAV2-GFP. Gene transfer and expression in the rat retina occurs quickly in 2 days and appears to peak within 2 weeks of gene delivery. In vivo retinal imaging may be a useful noninvasive tool to continuously monitor gene expression in the retina over time.
关键词： gene therapy,image analysis,retina
更新于2025-11-21 11:08:12
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Adeno-Associated Viral Gene Therapy for Inherited Retinal Disease
摘要： Inherited retinal diseases (IRDs) are a group of rare, heterogenous eye disorders caused by gene mutations that result in degeneration of the retina. There are currently limited treatment options for IRDs; however, retinal gene therapy holds great promise for the treatment of different forms of inherited blindness. One such IRD for which gene therapy has shown positive initial results is choroideremia, a rare, X-linked degenerative disorder of the retina and choroid. Mutation of the CHM gene leads to an absence of functional Rab escort protein 1 (REP1), which causes retinal pigment epithelium cell death and photoreceptor degeneration. The condition presents in childhood as night blindness, followed by progressive constriction of visual fields, generally leading to vision loss in early adulthood and total blindness thereafter. A recently developed adeno-associated virus-2 (AAV2) vector construct encoding REP1 (AAV2-REP1) has been shown to deliver a functional version of the CHM gene into the retinal pigment epithelium and photoreceptor cells. Phase 1 and 2 studies of AAV2-REP1 in patients with choroideremia have produced encouraging results, suggesting that it is possible not only to slow or stop the decline in vision following treatment with AAV2-REP1, but also to improve visual acuity in some patients.
关键词： gene therapy,choroideremia,AAV2-REP1,retina
更新于2025-09-23 15:23:52
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Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles
摘要： The marine microalga Chlamydomonas sp. JSC4 is a potential lutein source with high light tolerance. In this study, light intensity was manipulated to enhance cell growth and lutein production of this microalga. High lutein productivity (5.08 mg/L/d) was achieved under high light irradiation of 625 μmol/m2/s. Further increase in light intensity to 750 μmol/m2/s enhanced the biomass productivity to 1821.51 mg/L/d, but led to a decrease in lutein content. Under high light conditions, most carotenoids and chlorophyll contents decreased, while zeaxanthin and antheraxanthin contents increased. Inspection of gene expression profile shows that the lut1 and zep genes, responsible for lutein synthesis and flow of zeaxanthin into violaxanthin, respectively, were downregulated, while zeaxanthin biosynthesis gene crtZ was upregulated when the microalga was exposed to a high light intensity. This is consistent with the decrease in lutein content and increase in zeaxanthin content under high light exposure.
关键词： Lutein productivity,high light,Chlamydomonas sp.,gene expression
更新于2025-09-23 15:23:52
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A novel photosensitive dual-sensor for simultaneous detection of nucleic acids and small chemical molecules
摘要： Sensors that can rapidly and specifically detect nucleic acids and chemical molecules can revolutionize the diagnosis and treatment of diseases by allowing molecular-level informations to be used during the routine medicines. In this study, we demonstrated a novel dual-sensor that can be used to simultaneously detect any nucleic acids and chemical molecules whose binding aptamers can be found or synthesized. In the developed dual-sensor, the specifically designed PTG (a photosensitive azobenzene derivative carrying one photo-isomerizable azobenzene moiety, one threoninol terminal and one guanidinium terminal) molecules are introduced into the unwinding region of two T7 promoters, and two DNA bubbles are introduced upstream of the two T7 promoters. Without the target, the indicating gene in the dual-tensor would not be expressed since the binding with RNAPs (RNA polymerases) cannot melt the T7 promoter for the indicating gene due to the integration of the DNA double strands via the PTG molecules, manifesting the absence of the target nucleic acid and chemical molecule. While with the presence of the target nucleic acid and/or chemical molecule, the indicating gene would be expressed as the T7 promoter contained in the enlarged DNA bubble can be melted and transcribed by the bound RNAPs as the enlarged DNA bubble can help the separation of the two DNA strands, demonstrating the existence of target nucleic acid and/or chemical molecule.
关键词： Nucleic acids,DNA melting,Gene expression,DNA nanotechnology,Dual-sensor
更新于2025-09-23 15:23:52
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Novel Multimodality Imaging Markers for Image Guided Prostate and Breast Cancer Brachytherapy
摘要： Previous studies have revealed a significant association between SNPs found within the heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) gene and obesity. This study identified a novel 43-bp indel polymorphism in intron 1 of HS6ST3 in 1963 chickens from nine different breeds, and three genotypes, designated II, ID and DD, were observed. The frequency of the 'I' (0.62–0.87) allele was higher than that of the 'D' (0.13–0.38) allele. A total of 777 individuals of the Gushi-Anka F2 resource population were used for the analysis of associations according to growth traits, carcass traits, serum variables and meat quality traits. The results showed that the 43-bp indel polymorphism was significantly associated with the body weight at 4 and 6 weeks of age, chest depth at 4 and 12 weeks of age and shank girth at 12 weeks of age (P < 0.05). In terms of the carcass traits, the indel polymorphism was significantly associated with breast muscle weight, heart weight and leg weight (P < 0.05). These findings suggested that this indel polymorphism has the potential to become a new target for the marker-assisted selection of chicken growth and carcass traits.
关键词： insertion/deletion (indel),HS6ST3 gene,growth and carcass trait
更新于2025-09-23 15:23:52
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Dark-Adaptation Functions in Molecularly Confirmed Achromatopsia and the Implications for Assessment in Retinal Therapy Trials
摘要： PURPOSE. To describe the dark-adaptation (DA) functions in subjects with molecularly proven achromatopsia (ACHM) using refined testing conditions with a view to guiding assessment in forthcoming gene therapy trials. METHODS. The DA functions of nine subjects with ACHM were measured and compared with those of normal observers. The size and retinal location of the stimuli used to measure DA sensitivities were varied in four distinct testing condition sets, and the effect of altering these parameters assessed. RESULTS. In three of the four testing condition sets, achromats had significantly higher mean final thresholds than normal observers, whereas in the fourth condition set they did not. A larger, more central stimulus revealed the greatest difference between the final DA thresholds of achromat and normal subjects, and also demonstrated the slowest rate of recovery among the achromat group. CONCLUSIONS. In this, the largest study of DA functions in molecularly proven ACHM to date, we have identified optimal testing conditions that accentuate the relative difference between achromats and normal observers. These findings can help optimize DA testing in future trials, as well as help resolve the dichotomy in the literature regarding the normality or otherwise of DA functions in ACHM. Furthermore, the shorter testing time and less intense adaptation light used in these experiments may prove advantageous for more readily and reliably probing scotopic function in retinal disease, and be particularly valuable in the frequent post therapeutic assessments required in the context of the marked photophobia in ACHM.
关键词： rod monochromatism,dark adaptation,achromatopsia,rod vision,gene therapy
更新于2025-09-23 15:22:29