RESUMEN
The VisuFect-conjugated molecular beacon was developed for non-invasive visualization of microRNA34c in a living single mouse sperm.
Asunto(s)
Carbocianinas/análisis , Carbocianinas/química , MicroARNs/análisis , Espermatozoides/metabolismo , Animales , Supervivencia Celular , Fluorescencia , Células HEK293 , Humanos , Masculino , Ratones , MicroARNs/química , Microscopía Fluorescente , Análisis de la Célula IndividualRESUMEN
Current methods for delivering foreign genetic materials into mammalian cells are highly successful. However, these methods cannot be applied in oocyte or embryo systems due to their toxicity and low efficiency. Moreover, no satisfactory methods exist for delivering foreign genetic material without inducing physical damage to membranes. Here we developed an organic compound (VisuFect)-mediated small interfering RNA (siRNA) delivery method and evaluated this method in P19 cells and mouse zygotes. Oct4-siRNA conjugated VisuFect (Oct4-siRNA-VF) permeated the zona pellucida effectively and localized inside mouse zygotes without inducing membrane damage. Successful VisuFect-mediated delivery was further demonstrated by strong transcriptional repression of Oct4 expression by the delivered Oct4-siRNA, in addition to repressed embryonic development of mouse zygotes.
Asunto(s)
Técnicas de Transferencia de Gen , ARN Interferente Pequeño/administración & dosificación , Cigoto , Animales , Desarrollo Embrionario , Femenino , Ratones , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Factor 3 de Transcripción de Unión a Octámeros/genética , Embarazo , Zona PelúcidaRESUMEN
For adequate cancer therapy, newer imaging modalities with more specific ligands for unique targets are crucial. Underglycosylated mucin-1 (uMUC-1) antigen is an early marker of tumor development and is widely overexpressed on most tumors. A combination of nanotechnology with optical, radionuclide, and magnetic resonance (MR) imaging has great potential to improve cancer diagnosis and therapy. In this study, a multimodal nanoparticle imaging system was developed that can be used for optical, MR and positron emission tomography (PET) imaging. Cobalt ferrite magnetic nanoparticles surrounded by fluorescent rhodamine (designated MF) within a silica shell matrix were conjugated with an aptamer targeting uMUC-1 (designated MF-uMUC-1) and further labeled by (68)Ga (designated MFR-uMUC-1) with the help of a p-SCN-bn-NOTA chelating agent, resulting in single multimodal nanoparticles. The resultant nanoparticles are spherical and monodispersed, as revealed by transmission electron microscopy. The MFR-uMUC-1 nanoparticle showed specific and dose-dependent fluorescent, radioisotope and MR signals targeting BT-20 cells expressing uMUC-1. In vivo targeting and multimodal imaging in tumor-bearing nude mice also showed great specificity for targeting cancers with MFR-uMUC-1. The MFR-uMUC-1 probe could be used as a single multimodal probe to visualize cancer cells by means of optical, radionuclide and MR imaging.
Asunto(s)
Aptámeros de Nucleótidos , Mucina-1/genética , Imagen Multimodal , Neoplasias Experimentales/diagnóstico , Animales , Línea Celular Tumoral , Imagen por Resonancia Magnética , Ratones , Microscopía Electrónica de Transmisión , Neoplasias Experimentales/patología , Tomografía de Emisión de PositronesRESUMEN
A dual fluorophore-based color-tunable molecular beacon visualized the microRNA-294 expression-dependent color change in cells.
Asunto(s)
Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Regulación de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Imagen Molecular , Sondas de Oligonucleótidos/química , Animales , Células CHO , Diferenciación Celular , Color , Cricetinae , Cricetulus , Ratones , Microscopía Confocal , Neuronas/citologíaRESUMEN
Magnetic resonance imaging (MRI) offers great spatial resolution for viewing deep tissues and anatomy. We developed a self-assembling signal-on magnetic fluorescence nanoparticle to visualize intracellular microRNAs (miRNAs or miRs) during neurogenesis using MRI. The self-assembling nanoparticle (miR124a MR beacon) was aggregated by the incubation of three different oligonucleotides: a 3' adaptor, a 5' adaptor, and a linker containing miR124a-binding sequences. The T2-weighted magnetic resonance (MR) signal of the self-assembled nanoparticle was quenched when miR124a was absent from test tubes or was minimally expressed in cells and tissues. When miR124a was present in test tubes or highly expressed in vitro and in vivo during P19 cell neurogenesis, it hybridized with the miR124a MR beacon, causing the linker to detach, resulting in increased signal-on MRI intensity. This MR beacon can be used as a new imaging probe to monitor the miRNA-mediated regulation of cellular processes.
Asunto(s)
Espacio Intracelular/metabolismo , Imagen por Resonancia Magnética , MicroARNs/metabolismo , Sondas Moleculares , Neurogénesis , Diferenciación Celular/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Nanopartículas/ultraestructura , Neurogénesis/genéticaRESUMEN
A self-assembling magnetic resonance beacon was used to visualize the microRNA-1 expression-dependent change in magnetic resonance signal intensity.
Asunto(s)
MicroARNs/análisis , MicroARNs/química , Sondas de Oligonucleótidos/química , Animales , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Espectroscopía de Resonancia Magnética , Ratones , Nanopartículas/químicaRESUMEN
The development of gene delivery systems into embryos is challenging due to technical difficulties, delivery efficiency and toxicity. Here, we developed an organic compound (VisuFect)-mediated gene delivery system for zygotes. The VisuFect, which is hydrophilic and Cy5.5-labeled, was conjugated with poly(A) oligo (VFA). The VFA into CHO cells showed clathrin-mediated internalization and no toxicity. The VFA successfully penetrated through the zona pellucida of fertilized eggs of various species including pigs, zebrafish, drosophilas and mice. The experiment with VisuFect-mediated delivery of the miR34c inhibitor showed similar results with direct microinjection of the miR34c inhibitor by suppressing the development of zygotes up to the blastocyst stage. Noticeable features of the VisuFect will provide great benefits for further studies on gene function in sperms and embryos.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Oligonucleótidos/genética , Transfección/métodos , Cigoto/metabolismo , Animales , Células CHO , Carbocianinas/química , Células Cultivadas , Cricetinae , Cricetulus , Drosophila , Femenino , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Microinyecciones , Microscopía Confocal , Oligonucleótidos/administración & dosificación , Oligonucleótidos/química , Compuestos Orgánicos/química , Poli A/administración & dosificación , Poli A/química , Poli A/genética , Porcinos , Imagen de Lapso de Tiempo/métodos , Pez Cebra , Cigoto/crecimiento & desarrolloRESUMEN
NIRF imaging of carbon nanodots derived from tire soot was clearly visualized in glioma in vitro and in vivo.
Asunto(s)
Carbono/química , Fluorescencia , Glioma/química , Hollín/química , Animales , Línea Celular Tumoral , Microscopía Confocal , Ácido Nítrico/química , Tamaño de la Partícula , Ratas , Espectrometría de Fluorescencia , Propiedades de SuperficieRESUMEN
Using the excellent emission properties of carbon nanodots, we have developed an aptamer-conjugated imaging probe for targeting cancers.