RESUMEN
An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.
Asunto(s)
Vacunas contra el SIDA/inmunología , Infecciones por VIH/terapia , VIH-1/inmunología , Transfección/métodos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Animales , Reactores Biológicos , Células Dendríticas/inmunología , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , Humanos , Inmunogenicidad Vacunal/genética , Masculino , Células Madre Mesenquimatosas/inmunología , Ratones , Modelos Animales , Plásmidos/genética , Cultivo Primario de Células , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T/inmunología , Transfección/instrumentación , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400-800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.
Asunto(s)
Electroporación/métodos , Terapia Genética/métodos , Microfluídica/métodos , Transfección/métodos , Animales , Línea Celular Tumoral , Supervivencia Celular , Electroporación/instrumentación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Microfluídica/instrumentación , Plásmidos/genética , Transfección/instrumentación , TransgenesRESUMEN
Recently, stimuli-responsive gene carriers have been widely studied to overcome the extra- and intracellular barriers in cancer treatment. In this study, we modified polydopamine nanoparticles with low-molecular weight polyethylenimine (PEI1.8k) and polyethylene glycol-phenylboronic acid (PEG-PBA) to prepare pH-responsive gene carrier PDANP-PEI-rPEG. PBA and polydopamine could form pH-responsive boronate ester bonds. Non-responsive PDANP-PEI-nPEG and non-PEGylated PDANP-PEI were also studied as control. Both PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes remained stable in the pH environment of blood circulation or extracellular delivery (pHâ¯7.4) owing to the PEG modification. And after being internalized into endosomes, the boronate ester bonds could be cleaved. The pH responsive ability of PDANP-PEI-rPEG might facilitate complexes dissociation and gene release inside cells. The transfection level of PDANP-PEI-rPEG/DNA complexes was about 100 times higher than that of PDANP-PEI-nPEG/DNA complexes with the same mass ratios. Moreover, after NIR light irradiation at the power density of 2.6â¯W/cm2 for 20â¯min, the good photothermal conversion ability of PDANP resulted in quick endosomal escape. The transfection level of PDANP-PEI-rPEG/DNA complexes doubled, even higher than that of lipofectamine 2000/DNA complexes. This was also confirmed by Bafilomycin A1 inhibition test and CLSM observation. In response to the acidic pH within cancer cells and the NIR light irradiation, the PDANP-PEI-rPEG carrier could overcome multiple obstacles in gene delivery, which was promising for further application in gene therapy.
Asunto(s)
Técnicas de Transferencia de Gen , Indoles/química , Nanopartículas/química , Polímeros/química , Animales , Ácidos Borónicos/química , ADN/administración & dosificación , ADN/química , Endosomas , Femenino , Técnicas de Transferencia de Gen/instrumentación , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Macrólidos/farmacología , Ratones Desnudos , Peso Molecular , Polietilenglicoles/química , Polietileneimina/química , Espectroscopía Infrarroja por Transformada de Fourier , Transfección/instrumentación , Transfección/métodosRESUMEN
The common phenomenon that the nonviral vectors have much lower transfection efficiency in vivo than in vitro greatly restricts their further developments and applications. Possible reasons are lacking targeting ability, elimination by the reticuloendothelial system (RES), and insufficient nuclear transport. Here, a novel, flexible, and deformable polymer Fe@PEI-R12 (tLyp-1-NLS) is reported for shortening the gap between in vitro and in vivo gene transfection efficiency. The amorphous network structure Fe@PEI with deformation ability acquired by coordination cross-linking of Fe3+ and low-molecular-weight polyethylenimine (LMW-PEI) constructs the core and serves as the gene reservoir, and it can squeeze out through RES filter holes when trapped in the spleen. The bifunctional peptide R12 provided tumor targeting and enhanced nuclear delivery ability. Additionally, the Fe3+ from Fe@PEI-R12 could trigger endogenous hydrogen peroxide (H2O2) decomposition to produce O2, thereby reducing the adverse effects of tumor hypoxia. It is demonstrated that the Fe@PEI-R12/pDNA complexes could pass through membrane filters, subsequently achieving long circulation time, and Fe@PEI-R12 had a tendency to accumulate in tumor tissue and mediate pGL3-control expression. Therefore, the multifunctional nanoplatform has the potential for effective in vivo gene delivery.
Asunto(s)
Compuestos Férricos/química , Terapia Genética/instrumentación , Nanoestructuras/química , Neoplasias/terapia , Péptidos/administración & dosificación , Péptidos/química , Transfección/métodos , Animales , Terapia Genética/métodos , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias/genética , Péptidos/genética , Polietileneimina/química , Transfección/instrumentaciónRESUMEN
Delivery of macromolecular nucleotides into the living cells holds a great promise for the development of new therapeutics. However, its abilities for adoptive immunotherapy, cell reprogramming, and primary cell transfection have been long-term hindered by the lack of a system that can locally deliver engineered therapeutic nucleotides (e.g., plasmids, siRNAs, miRNAs) without causing any side effects. In this chapter, the performance of a novel 3D nanoelectroporation system (3D NEP) is highlighted in three scenarios-adoptive immunotherapy, cell reprogramming, and adult mouse primary cardiomyocyte transfection. Detailed protocols were given to introduce the 3D NEP system assembly, as well as their applications in (1) natural killer (NK) cells transfection by delivery of chimeric antigen receptor (CAR) plasmids; (2) mouse embryonic fibroblasts transfection with OSKM factors; and (3) miR-29b molecular beacon (BMs) delivery into primary cardiomyocytes for interrogating the side effect of miR-29b-assisted treatment.
Asunto(s)
Electroporación/instrumentación , Fibroblastos/citología , Células Asesinas Naturales/citología , Miocitos Cardíacos/citología , Nucleótidos/administración & dosificación , Animales , Células Cultivadas , Técnicas de Reprogramación Celular/instrumentación , Técnicas de Reprogramación Celular/métodos , Fibroblastos/química , Humanos , Inmunoterapia Adoptiva/instrumentación , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/química , Ratones , Miocitos Cardíacos/química , Nanotecnología , Transfección/instrumentación , Transfección/métodosRESUMEN
CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit genomic DNA for studying biology, pathogenesis, and molecular basis of treatment in malignant B cells. Unfortunately, malignant B cells are extremely difficult to transfect by most traditional methods. In this chapter, we describe the use of the Nucleofector™ Technology-based electroporation system with optimized transfection conditions for generating a malignant B cell model, JEKO-1, with ROR1-gene knockout via CRISPR-Cas9 technology.
Asunto(s)
Electroporación/instrumentación , Técnicas de Inactivación de Genes/instrumentación , Linfoma de Células B/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Humanos , Mutación con Pérdida de Función , Transfección/instrumentaciónRESUMEN
Electroporation is a common method of transfection due to its relatively low risk and high transfection efficiency. The most common method of electroporation is bulk electroporation which is easily performed on large quantities of cells yet results in variable levels of viability and transfection efficiency across the population. Localized electroporation is an alternative that can be administered on a similar scale but results in much more consistent with higher quality transfection and higher cell viability. This chapter discusses the creation and use of a simple and cost-effective device using porous membrane for performing localized electroporation.
Asunto(s)
Electroporación/instrumentación , Proteínas Fluorescentes Verdes/genética , Técnicas Analíticas Microfluídicas/instrumentación , Transfección/instrumentación , Línea Celular Tumoral , Supervivencia Celular , Electroporación/métodos , Células HeLa , Humanos , Dispositivos Laboratorio en un Chip , Plásmidos/genética , Transfección/métodosRESUMEN
The CRISPR/Cas9 system is transforming many biomedical disciplines, including cancer research. Through its flexible programmability and efficiency to induce DNA double strand breaks it has become straightforward to introduce cancer mutations into cells in vitro and/or in vivo. However, not all mutations contribute equally to tumorigenesis and distinguishing essential mutations for tumor growth and survival from biologically inert mutations is cumbersome. Here we present a method to screen for the functional relevance of mutations in high throughput in established cancer cell lines. We employ the CRISPR/Cas9 system to probe cancer vulnerabilities in a colorectal carcinoma cell line in an attempt to identify novel cancer driver mutations. We designed 100 high quality sgRNAs that are able to specifically cleave mutations present in the colorectal carcinoma cell line RKO. An all-in-one lentiviral library harboring these sgRNAs was then generated and used in a pooled screen to probe possible growth dependencies on these mutations. Genomic DNA at different time points were collected, the sgRNA cassettes were PCR amplified, purified and sgRNA counts were quantified by means of deep sequencing. The analysis revealed two sgRNAs targeting the same mutation (UTP14A: S99delS) to be depleted over time in RKO cells. Validation and characterization confirmed that the inactivation of this mutation impairs cell growth, nominating UTP14A: S99delS as a putative driver mutation in RKO cells. Overall, our approach demonstrates that the CRISPR/Cas9 system is a powerful tool to functionally dissect cancer mutations at large-scale.
Asunto(s)
Sistemas CRISPR-Cas/genética , Neoplasias Colorrectales/genética , Análisis Mutacional de ADN/métodos , Edición Génica/métodos , Biblioteca Genómica , Línea Celular Tumoral , Clonación Molecular/métodos , Análisis Mutacional de ADN/instrumentación , Vectores Genéticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lentivirus/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/aislamiento & purificación , Transfección/instrumentación , Transfección/métodosRESUMEN
For gene therapy to work in vivo, nucleic acids need to reach the target cells without causing major side effects to the patient. In many cases the gene only has to reach a subset of cells in the body. Therefore, targeted delivery of genes to the desired tissue is a major issue in gene delivery. Many different possibilities of targeted gene delivery have been studied. A physical approach to target nucleic acids and other drugs to specific regions in the body is the use of ultrasound and microbubbles. Microbubbles are gas filled spheres with a stabilizing lipid, protein, or polymer shell. When these microbubbles enter an ultrasonic field, they start to oscillate. The bubbles' expansion and compression are inversely related to the pressure phases in the ultrasonic field. When microbubbles are exposed to high-intensity ultrasound the microbubbles will eventually implode and fragment. This generates shockwaves and microjets which can temporarily permeate cell membranes and blood vessels. Nucleic acids or (non)viral vectors can as a result gain direct access to either the cytoplasm of neighboring cells, or extravasate to the surrounding tissue. The nucleic acids can either be mixed with the microbubbles or loaded on the microbubbles. Nucleic acid loaded microbubbles can be obtained by coupling nucleic acid-containing particles (i.e., lipoplexes) to the microbubbles. Upon ultrasound-mediated implosion of the microbubbles, the nucleic acid-containing particles will be released and will deliver their nucleic acids in the ultrasound-targeted region.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de la radiación , Ácidos Nucleicos/metabolismo , Transfección/métodos , Animales , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Humanos , Lípidos/química , Microburbujas , Ácidos Nucleicos/genética , Transfección/instrumentación , Ondas UltrasónicasRESUMEN
Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.
Asunto(s)
Autofagia/genética , Técnicas de Silenciamiento del Gen/métodos , ARN Interferente Pequeño/metabolismo , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Técnicas de Silenciamiento del Gen/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Asociadas a Microtúbulos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transfección/instrumentación , Transfección/métodosRESUMEN
Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia Linfocítica Crónica de Células B/genética , Proteína p53 Supresora de Tumor/genética , Alelos , Análisis Mutacional de ADN/instrumentación , Análisis Mutacional de ADN/métodos , Genes Reporteros/genética , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/patología , Mutación , Células Neoplásicas Circulantes/patología , Saccharomyces cerevisiae/genética , Transfección/instrumentación , Transfección/métodosRESUMEN
Cellular senescence, a state of permanent growth arrest, is an important mechanism preventing the propagation of damaged cells. It suppresses cancer development in premalignant lesions in response to activated oncogenes and in tumors following therapy. The presence of senescent cells in premalignant lesions and tumors is controlled by the immune system. The ability to identify and quantify senescent cells more efficiently in vivo is necessary in order to evaluate the effect of these cells on tumorigenesis and cancer therapy. Through combining senescent-associated beta-galactosidase staining with ImageStream X analysis, we have developed an effective method to identify and quantify senescent cancer cells in vivo.
Asunto(s)
Senescencia Celular/inmunología , Citometría de Flujo/métodos , Neoplasias/patología , Coloración y Etiquetado/métodos , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos , Citometría de Flujo/instrumentación , Galactosa/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Desnudos , Neoplasias/inmunología , Coloración y Etiquetado/instrumentación , Transfección/instrumentación , Transfección/métodos , beta-Galactosidasa/metabolismoRESUMEN
This chapter describes the potential use of viral-mediated gene transfer in the central nervous system for genome editing in the context of Huntington's disease. Here, we provide protocols that cover the design of various genome editing strategies, the cloning of CRISPR/Cas9 elements into lentiviral vectors, and the assessment of cleavage efficiency, as well as potential unwanted effects.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Terapia Genética/métodos , Proteína Huntingtina/genética , Enfermedad de Huntington/terapia , Animales , Encéfalo/patología , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Clonación Molecular/métodos , Modelos Animales de Enfermedad , Edición Génica/instrumentación , Terapia Genética/instrumentación , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Lentivirus/genética , Ratones , Ratones Transgénicos , Mutación , ARN Guía de Kinetoplastida/genética , Técnicas Estereotáxicas/instrumentación , Transfección/instrumentación , Transfección/métodosRESUMEN
Mitochondrial autophagy or mitophagy is a process that selectively degrades mitochondria via autophagy. It is believed that mitophagy degrades damaged or unnecessary mitochondria and is important for maintaining mitochondrial homeostasis. To date, it is known that several stimuli can induce mitophagy. However, some of these stimuli (including iron depletion, hypoxia, and nitrogen starvation) induce mild mitophagy, which is difficult to detect by measuring the decrease in mitochondrial mass. Recently, we have successfully detected mitophagy induced under these conditions using mito-Keima as a reporter. In this chapter, we describe the protocols for induction and detection of iron depletion- and hypoxia-induced mitophagy using the mito-Keima-expressing cells.
Asunto(s)
Hipoxia de la Célula , Hierro/metabolismo , Mitocondrias/metabolismo , Mitofagia/efectos de los fármacos , Transfección/métodos , Animales , Canales de Calcio/química , Canales de Calcio/genética , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Fibroblastos , Vectores Genéticos/genética , Células HeLa , Humanos , Quelantes del Hierro/farmacología , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Lisosomas/metabolismo , Ratones , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Mitocondrias/efectos de los fármacos , Retroviridae/genética , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/genética , Transfección/instrumentaciónRESUMEN
We developed a tissue suction-mediated transfection method (suction method) as a relatively reliable and less invasive technique for in vivo transfection. In this study, we determined hepatic transgene expression characteristics in the mouse liver, using a suction device, collecting information relevant to gene therapy and gene functional analysis by the liver suction method. To achieve high transgene expression levels, we developed a suction device with four holes (multiple hole device) and applied it to the larger portion of the left lateral lobe of the mouse liver. Hepatic transfection with physical stimuli was potentially controlled by activator protein-1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We examined the spatial distribution of transgene expression in the suctioned lobe by 2-dimensional imaging with histochemical staining and 3-dimensional multicolor deep imaging with tissue clearing methods. Through monitoring spatial distribution of transgene expression, the liver suction method was used to efficiently transfect extravascular hepatocytes in the suction-deformable upper lobe of the liver. Moreover, long-term transgene expression, at least 14 d, was achieved with the liver suction method when cytosine-phosphate-guanine (CpG)-free plasmid DNA was applied.
Asunto(s)
Hígado/metabolismo , Transfección/instrumentación , Transgenes , Animales , ADN , Femenino , Genes fos , Genes jun , Luciferasas/sangre , Luciferasas/genética , Luciferasas/metabolismo , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Plásmidos , Succión , Factor de Transcripción AP-1/metabolismo , Transfección/métodosRESUMEN
In the present study, Fe3O4/chitosan biopolymer grafted to a novel organosilane modified 5-amino-1H-tetrazole, a kind of drug intermediate, was successfully synthesized by chemical modification technique and evaluated as a high potential carrier of gene delivery. The loading capacity was evaluated, and in vitro release of nanocarrier was assessed using the dialysis method. The transfection efficiency of plasmid was optimal at an N/P ratio of 3. The chemically modified chitosan showed no inherent toxicity toward the cells. The synthesized nanocarrier had enhanced release of the plasmid at physiological pHâ¯7.4. The N-functionalized magnetic chitosan nanocarrier demonstrated its efficacy in the enhancement of gene expression in the HECK-293T cell line. Therefore, the novel magnetic N-functionalized chitosan showed promise as a highly efficient gene carrier with potential applications in cancer therapy.
Asunto(s)
Quitosano/química , Nanopartículas de Magnetita/química , Tiadiazoles/química , Transfección/métodos , Supervivencia Celular/efectos de los fármacos , Óxido Ferrosoférrico/química , Células HEK293 , Humanos , Nanopartículas de Magnetita/toxicidad , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Plásmidos/genética , Espectroscopía Infrarroja por Transformada de Fourier , Transfección/instrumentaciónRESUMEN
While luminescent reporter gene assays allow for a rapid and relatively interference free assessment of the activation state of a luminescent reporter, fluorescent reporters do not. They suffer from artifacts such as compound fluorescence and cellular debris which makes the assessment of whole well fluorescence signals difficult. However, the use of high-content screening allows for the isolation of individual cells, segmentation and thus enables the screener to utilize fluorescent reporters to assess the activation state of such a high-content reporter on a cell by cell level, thus minimizing artifacts. Here we discuss the use of such a high-content reporter that enables screening for compounds useful for HIV reactivation on Jurkat cells with high-content screening.
Asunto(s)
Bioensayo/métodos , Genes Reporteros/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Transfección/métodos , Fosfatasa Alcalina/química , Fosfatasa Alcalina/genética , Bioensayo/instrumentación , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , VIH/fisiología , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Microscopía Intravital/instrumentación , Microscopía Intravital/métodos , Células Jurkat , Luciferasas/química , Luciferasas/genética , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Transfección/instrumentación , Activación Viral/genética , beta-Galactosidasa/química , beta-Galactosidasa/genéticaRESUMEN
Spatio-temporal organization of the cell nucleus adapts to and regulates genomic processes. Microscopy approaches that enable direct monitoring of specific chromatin sites in single cells and in real time are needed to better understand the dynamics involved. In this chapter, we describe the principle and development of ANCHOR, a novel tool for DNA labelling in eukaryotic cells. Protocols for use of ANCHOR to visualize a single genomic locus in eukaryotic cells are presented. We describe an approach for live cell imaging of a DNA locus during the entire cell cycle in human breast cancer cells.
Asunto(s)
ADN/química , Sitios Genéticos/genética , Microscopía Intravital/métodos , Imagen Molecular/métodos , Coloración y Etiquetado/métodos , Ciclo Celular/genética , Humanos , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Microscopía Intravital/instrumentación , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Células MCF-7 , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Imagen Molecular/instrumentación , Transfección/instrumentación , Transfección/métodos , Transgenes/genéticaRESUMEN
Delivery of large and structurally complex target molecules into cells is vital to the emerging areas of cellular modification and molecular therapy. Inadequacy of prevailing in vivo (viral) and in vitro (liposomal) gene transfer methods for delivery of proteins and a growing diversity of synthetic nanomaterials has encouraged development of alternative physical approaches. Efficacy of injury/diffusion-based delivery via shear mechanoporation is largely insensitive to cell type and target molecule; however, enhanced flexibility is typically accompanied by reduced gene transfer effectiveness. We detail a method to improve transfection efficiency through coordinated mechanical disruption of the cell membrane and electrophoretic insertion of DNA to the cell interior. An array of micromachined nozzles focuses ultrasonic pressure waves, creating a high-shear environment that promotes transient pore formation in membranes of transmitted cells. Acoustic Shear Poration (ASP) allows passive cytoplasmic delivery of small to large nongene macromolecules into established and primary cells at greater than 75% efficiency. Addition of an electrophoretic action enables active transport of target DNA molecules to substantially augment transfection efficiency of passive mechanoporation/diffusive delivery without affecting viability. This two-stage poration/insertion method preserves the compelling flexibility of shear-based delivery, yet substantially enhances capabilities for active transport and transfection of plasmid DNA.
Asunto(s)
Electroforesis/métodos , Técnicas de Transferencia de Gen , Transfección/métodos , Ondas Ultrasónicas , Línea Celular , Permeabilidad de la Membrana Celular , ADN/administración & dosificación , Difusión , Electroforesis/instrumentación , Electroporación , Humanos , Sustancias Macromoleculares/administración & dosificación , Transfección/instrumentaciónRESUMEN
RNA interference (RNAi) is a normal physiological mechanism in which a short effector antisense RNA molecule regulates target gene expression. It is a powerful tool to silence a particular gene of interest in a sequence-specific manner and can be used to target against various molecular pathways in esophageal adenocarcinoma by designing RNAi targeting key pathogenic genes. RNAi-based therapeutics against esophageal adenocarcinoma can be developed using different strategies including inhibition of overexpressed oncogenes, blocking cell division by interfering cyclins and related genes or enhancing apoptosis by suppressing anti-apoptotic genes. In addition, RNAi against multidrug resistance genes or chemo-resistance targets may provide promising cancer therapeutic options. Here, we describe RNAi technology using MET, a proto-oncogene in esophageal adenocarcinoma cells, as a model target. Lentiviral particles expressing MET shRNA was used to silence MET genes. Then, Western blot analysis was performed to confirm MET knockdown.