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Cloning Full-Length cDNAs from Vascular Tissues and Cells by Rapid Amplification of cDNA Ends (RACE) and RT-PCR

The isolation of full-length cDNAs remains a frequent task undertaken in many laboratories. A full-length cDNA is often desirable for one of the following purposes: 1) to complete the sequence of a partial cDNA cloned by library screenings or the yeast one- or two-hybrid system; 2) to derive the cDNA se ...

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The Use of Differential mRNA Display (DDRT-PCR) to Identify Genes Differentially Expressed in Normal and Diseased Vascular Cells

In 1992 a new approach for identifying differentially expressed genes was described by Liang and Pardee (1). Their method allowed the simultaneous differential display of mRNA from two or more cell types by means of the polymerase chain reaction (PCR). In a subsequent study demonstrating the t ...

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Primer Extension Analysis to Map Transcription Start Sites of Vascular Genes

The principle of the technique presented in this chapter is illustrated in Fig. 1. As with S1 mapping or riboprobe mapping, this technique can be used to determine precisely the start site of transcription of a mRNA sequence (1–3). Since this technique is relatively easier than other techniques, it is ...

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Use of Liposome-Mediated DNA Transfection to Determine Promoter Activity in Smooth Muscle Cells

The transfer and expression of DNA plasmids containing promoter fragments of heterologous genes linked to reporter cDNAs in mammalian cells has become an invaluable technique for studying the regulation of gene expression. Several reporter genes such as luciferase, β-galactosi ...

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Nuclear Run-On Assay to Study Gene Transcription in Vascular Cells

An accurate assessment of gene transcription is important for understanding the mechanism of gene expression. The nuclear run-on assay measures the relative in situ transcription rate of specific genes in intact nuclei (1,2). It provides information on the synthesis of a specific gene th ...

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Electromobility Shift Analysis (EMSA) Applied to the Study of NF-kappa B Binding Interactions in Vascular Smooth Muscle Cells

The nuclear factor-kappa B (NFκB) family of transcription factors has emerged as a signaling pathway that figures prominently in a cell’s initial response to a plethora of inflammatory stimuli. Modified lipids, oxidative stress, bacterial endotoxins, growth factors and cytokines, s ...

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Measurement of Gene Expression in the Vascular Wall by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

The developments of the polymerase chain reaction (PCR) analysis (1–3) and quantitative PCR by Gilliland (4) has provided researchers with a unique tool to analyze the expression of various genes in very small amounts of tissue samples. The sensitivity of PCR allows measurement of the expres ...

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Northern Blot Analysis to Quantify Gene Expression in Vascular Diseases

To understand the molecular mechanisms of vascular diseases, studies are designed to process gene function from the discovery of relevant genes to examination of their expression and association with disease. Once a gene has been cloned, Northern blot analysis can directly monitor the g ...

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Localization of Gene Expression in Human Atherosclerotic Lesions by In SituHybridization

The technique of in situ hybridization was developed in the late 1960s (1) and is based on the ability of a single-stranded sequence of nucleotides to hybridize specifically to a complementary sequence to form stable double-stranded duplexes or hybrids. Incorporating a readily detectab ...

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Detection of Apoptosis in Atherosclerosis and Restenosis by Terminal dUTP Nick-End Labeling (TUNEL)

Kerr and Wyllie (1) have introduced the term apoptosis to separate this special form of cell death from necrosis. When a cell receives a signal to die an apoptotic death, it goes through a series of morphological changes that can be easily observed with the light microscope. Starting from shrinkage of t ...

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RNase Protection Assays for Quantitation of Gene Expression in Vascular Tissue

Ribonuclease protection assay (RPA) is a sensitive solution hybridization method for quantitation of specific RNAs (1–3). The method is based on the ability of single-strand specific ribonuclease to degrade single-stranded RNA while leaving intact fragments of labeled antisense ...

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Molecular Assessment of the Phenotypic Changes Associated with Smooth Muscle Cells Using Two-Dimension Electrophoresis and Microsequence Analysis

Phenotypic modulation of arterial smooth muscle cells (SMC) is essential for the evolution of atheromatosis and restenosis after angioplasty (1). During these pathological phenomena, SMC express numerous genes such as those responsible for cell migration and proliferation (2). T ...

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In Vitro Detection of Apoptosis in Isolated Vascular Cells

Apoptosis is a programmed cell death process in which surplus or damaged cells are eliminated through a highly regulated procedure. The first description of apoptosis relied upon morphological differences among apoptotic, necrotic, and healthy cells (1). Indeed, there are usually a nu ...

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Generation of Recombinant Adeno-Associated Viruses for Delivery of Genes into Vascular Cells

A variety of delivery systems, both viral and nonviral, have been employed to genetically modify vascular endothelial cells and smooth muscle cells (SMC) in vitro and by direct in vivo gene transfer into the vessel wall. The most recent addition to gene delivery technology for the vasculature has ...

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Simple Methods for Preparing Recombinant Adenoviruses for High-Efficiency Transduction of Vascular Cells

Adenoviruses are icosahedral viruses 70–90 nm in diameter with a double-stranded, linear DNA genome of approx 36 kb. They are widely utilized in gene transfer protocols owing to their relative ease of genetic manipulation, ability to grow to high titres (109–1012 plaque forming units /mL), and t ...

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High-Efficiency and Low-Toxicity Adenovirus-Assisted Endothelial Transfection

Cultured endothelial cells have provided a powerful tool for discovery of the molecular regulators of a range of vascular processes from angiogenesis to fibrinolysis (1). Yet, the utility of genetic manipulation of endothelial culture systems to dissect critical intracellular si ...

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Hemagglutinating Virus of Japan Liposome-Mediated Gene Delivery to Vascular Cells

Since the first report of in vivo direct gene transfer to the vessel wall in 1990 (1) several vectors, such as adenovirus, liposomes, and adeno-associated virus have been employed to introduce foreign genes to the vascular tissue in vivo. Hemagglutinating virus of Japan (HVJ, Sendai virus), a memb ...

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Gene Transfer in Vascular Cells Using an Engineered Na-H Exchanger (NHE1) as a Selectable Marker

The techniques of gene transfer using transfection via electroporation, CaPO4, or cationic lipids rely on selectable markers because of the low efficiency of this approach. Selectable markers range from fluorescent molecules to a variety of cytotoxic compounds, with the most common ...

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Embryonal Stem (ES) Cell-Derived Macrophages: A Cellular System that Facilitates the Genetic Dissection of Macrophage Function

The monocyte/macrophage (M�) contributes to atherosclerotic lesion initiation and progression through a variety of interactions with cells of the artery wall that depend on the elucidation of a host of cytokines and growth factors by cells residing in the intima. The number and complexity ...

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Delivery of Recombinant Adenoviruses to Human Saphenous Vein

The human saphenous vein is the most commonly used conduit for coronary artery bypass grafting owing to its ready availability, ease of harvesting, and favorable surgical handling (1). However, it suffers from a progressive decline in patency, resulting in a graft failure rate of 50% after 10 yr (2, ...

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