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Engineering Novel Molecular Beacon Constructs to Study Intracellular RNA Dynamics and Localization

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Genomics, Proteomics & Bioinformatics 2017年第5期15卷 279-286页

作者： Zhao Ma Xiaotian Wu christopher J.Krueger Antony K.Chen Department of Biomedical Engineering College of Engineering Peking University Beijing 100871 China Wallace H coulter Department of Biomedical Engineering Georgia Institute of Technology Atlanta GA 30332 USA

With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss tech- niques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons （MBs）, a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells. With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of RNA functions and activities.

关键词： Y-O-methyl RNA Phosphorothioate Molecular beacon RNA dynamics Single-molecule RNAimaging

Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms

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Genomics, Proteomics & Bioinformatics 2019年第2期17卷 119-128页

作者： Xiaotian Wu Shiqi Mao Yachen Ying christopher J.Krueger Antony K. Chen Department of Biomedical Engineering College of Engineering Peking University Beijing 100871 China School of Life Sciences Peking University Beijing 100871 China Wallace H coulter Department of Biomedical Engineering Georgia Institute of Technology Atlanta GA 30332 USA

Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states,the requirement for cell fix-ation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activ-ities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review,we describe currently-available approaches for imaging single genomic loci in cells,with special focus on clus-tered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition,we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches,and potential solutions to address these challenges. We anticipate that,with continued refinement of CRISPR-based imaging techniques,significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.

关键词： CRISPR Cas9 dCas9 Genomic imaging sgRNA

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

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Protein & Cell 2021年第1期12卷 39-56页

作者： Bingzhou Han Yage Zhang Xuetong Bi Yang Zhou christopher JKrueger Xinli Hu Zuoyan Zhu Xiangjun Tong Bo Zhang Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education Peking University Genome Editing Research CenterCollege of Life SciencesPeking UniversityBeiing 100871China School of Biology and Biological Engineering South China University of TechnologyGuangzhou 510006China Department of Biomedical Engineering College of EngineeringPeking UniversityBeijing 100871China Wallace H.coulter Department of Biomedical Engineering Georgia Institute of Techno logy and EmoryAtlantaGA 33032USA Institute of Molecular Medicine Peking UniversityBejing 100871China

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene ***,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been *** we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas *** demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage *** then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent *** introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible *** named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 ***,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA *** system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

关键词： CRISPR/Cas conditional knockout allele labeling conditional rescue minicircle DNA

Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation

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Protein & Cell 2018年第7期9卷 640-651页

作者： Na Qu Zhao Ma Mengrao Zhang Muaz N. Rushdi christopher J. Krueger Antony K. Chen Department of Biomedical Engineering College of Engineering Peking University Beijing 100871 China Wallace H coulter Department of Biomedical-Engineering Georgia Institute of Technology Atlanta GA 30332 USA

We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-medi- ated Gag assembly at the plasma membrane. Conse- quently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demon- strate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endo- somesllysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autop- hagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus （MLV） assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by non- specific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possi- bility that similar regulations can occur in other biolog- ical contexts.

关键词： microRNA Gag protein autophagy

A bio-instructive parylene-based conformal coating suppresses thrombosis and intimal hyperplasia of implantable vascular devices

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Bioactive Materials 2023年第10期28卷 467-479页

作者： Dake Hao Jonathan Lin Ruiwu Liu christopher Pivetti Kaeli Yamashiro Linda M.Schutzman Junichiro Sageshima Mimmie Kwong Nataliya Bahatyrevich Diana L.Farmer Misty D.Humphries Kit S.Lam Alyssa Panitch Aijun Wang Department of Surgery School of MedicineUniversity of California DavisSacramentoCA95817United States Institute for Pediatric Regenerative Medicine Shriners Hospitals for ChildrenSacramentoCA95817United States Department of Biochemistry and Molecular Medicine School of MedicineUniversity of California DavisSacramentoCA95817United States Wallace H.coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory UniversityAtlant1GA30332United States Department of Biomedical Engineering University of California DavisDavisCA95616United States

Implantable vascular devices are widely used in clinical treatments for various vascular diseases. However, current approved clinical implantable vascular devices generally have high failure rates primarily due to their surface lacking inherent functional endothelium. Here, inspired by the pathological mechanisms of vascular device failure and physiological functions of native endothelium, we developed a new generation of bioactive parylene (poly(p-xylylene))-based conformal coating to address these challenges of the vascular devices. This coating used a polyethylene glycol (PEG) linker to introduce an endothelial progenitor cell (EPC) specific binding ligand LXW7 (cGRGDdvc) onto the vascular devices for preventing platelet adhesion and selectively capturing endogenous EPCs. Also, we confirmed the long-term stability and function of this coating in human serum. Using two vascular disease-related large animal models, a porcine carotid artery interposition model and a porcine carotid artery-jugular vein arteriovenous graft model, we demonstrated that this coating enabled rapid generation of self-renewable “living” endothelium on the blood contacting surface of the expanded polytetrafluoroethylene (ePTFE) grafts after implantation. We expect this easy-to-apply conformal coating will present a promising avenue to engineer surface properties of “off-the-shelf” implantable vascular devices for long-lasting performance in the clinical settings.

关键词： Parylene coating Integrin ligand Thrombosis Intimal hyperplasia Implantable vascular devices

Characterizing Thermal Augmentation of Convection-Enhanced Drug Delivery with the Fiberoptic Microneedle Device

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Engineering 2015年第3期1卷 344-350页

作者： R.Lyle Hood Rudy T.Andriani Tobias E.Ecker John L.Robertson christopher G.Rylander Department of Nanomedicine Houston Methodist Research Institute Department of Mechanical Engineering Virginia Tech School of Biomedical Engineering Virginia Tech Department of Mechanical Engineering University of Texas

Convection-enhanced delivery （CED） is a promising technique leveraging pressure-driven flow to increase penetration of infused drugs into interstitial spaces. We have developed a fiberoptic microneedle device for inducing local sub-lethal hyperthermia to further improve CED drug distribution volumes, and this study seeks to quantitatively characterize this approach in agarose tissue phantoms. Infusions of dye were conducted in 0.6% （w/w） agarose tissue phantoms with isothermal conditions at 15 ℃, 20℃, 25 ℃, and 30 ℃. Infusion metrics were quantified using a custom shadowgraphy setup and image- processing algorithm. These data were used to build an empirical predictive temporal model of distribution volume as a function of phantom temperature. A second set of proof- of-concept experiments was conducted to evaluate a novel fiberoptic device capable of generating local photothermal heating during fluid infusion. The isothermal infusions showed a positive correlation between temperature and distribution volume, with the volume at 30℃ showing a 7-fold increase at 100 min over the 15 ℃ isothermal case. Infusions during photothermal heating （1064 nm at 500 mW） showed a similar effect with a 3.5-fold increase at 4 h over the control （0 mW）. These results and analyses serve to provide insight into and characterization of heat-mediated enhancement of volumetric dispersal.

关键词： near-infrared laser thermochemotherapy agarose photothermal heating micro-catheter malignant glioma