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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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An In Vivo Angiogenesis Assay to Study Positive and Negative Regulators of Neovascularization

The formation of new blood vessels from existing blood vessels has been referred to as angiogenesis to distinguish the process from de novo embryonic vessel formation or vasculogenesis (1). This chapter will describe an in vivo assay to measure angiogenesis. There are several important re ...

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Efficient Liposome-Mediated Gene Transfer to Rabbit Carotid Arteries In Vivo

Methods for gene delivery in vivo vary dramatically in their relative efficiencies. The most efficient to date involve the use of recombinant viruses, most notably adenoviruses, adeno-associated viruses (AAVs), or retroviruses (see Chapters 32 and 34). The creation of these recombina ...

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Gene Delivery to Rabbit Arteries Using the Collar Model

Emerging knowledge of molecular pathology of vascular diseases provides new targets for vascular gene therapy. Sufficient expression of a gene of interest in the vessel wall can be achieved using either extravascular or intravascular gene delivery approaches (1). Plasmid DNA, trans ...

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Local Gene Delivery of Recombinant Adenoviruses to the Rat Carotid Artery In Vivo

A number of animal models are available to investigators wishing to study the use of gene transfer to prevent neointimal formation after vascular injury. The majority are models of primary vascular injury rather than the human situation, where recurrence of a stenosis occurs in an abnormal bl ...

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Delivery of Antisense Oligonucleotides to the Vascular Wall

Antisense oligonucleotides are short segments of synthetic DNA designed to contain sequences of bases complementary to the DNA or RNA of a particular target gene of interest. By binding to the target, the antisense oligonucleotides can prevent translation of the gene into protein via diff ...

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Hemostasis: Components and Processes

Hemostasis is a host defense mechanism that protects the integrity of the vascular system after tissue injury. It works in conjunction with other inflammatory, immune, and repair mechanisms to produce a coordinated response. Hemostatic systems are generally quiescent, but followi ...

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Isolation of DNA and RNA

Blood samples for most coagulation tests are collected into 3.8% trisodium citrate in a ratio of 1 part anticoagulant to 9 parts blood. Whole-blood samples for DNA isolation can be stored at −50�C and the DNA prepared at a later stage. A more convenient method requiring less freezer space is to store buffy c ...

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Amplification of DNA and RNA by PCR

The polymerase chain reaction (PCR) has revolutionized many areas of medicine, including hemostasis. Although this volume is not devoted to PCR, many of the chapters employ the technique at some point to amplify specific DNA or RNA sequences. This chapter, therefore, provides a brief outline ...

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Solid-Phase Sequencing of Biotinylated PCR Products with Streptavidin-Coated Magnetic Beads

A novel approach to generating high-quality single-stranded DNA involves solid-phase sequencing. A biotin group is incorporated at the 5′-end of one of the amplification primers and as a result becomes incorporated into PCR product during the amplification reaction. The DNA can then be im ...

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Detection of DNA by Silver Staining

Silver stains are widely used for the detection of both proteins and nucleic acids in acrylamide gels or on various membranes. They have several advantages over conventional staining methods, including the ability to detect very small amounts of material, while avoiding the hazards of oth ...

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Promoter Studies in Hemostasis

In recent years the technique of gene cloning, coupled with the application of polymerase chain reaction (PCR)-based procedures, has greatly facilitated the production of cloned genomic material encompassing the putative promoter regions of genes involved in the hemostatic proc ...

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Screening for Mutations in DNA by Single-Stranded Conformation Polymorphism (SSCP) Analysis

A single-stranded DNA fragment may adopt several different conformations (conformers) which affect its electrophoretic mobility. The mobility of single-stranded DNA in a nondenaturing gel is dependent on both fragment length and secondary structure, which is sequence-depen ...

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Detection of Mutations Causing Hemophilia A Using an In Vitro Coupled Transcription and Translation System

Mutation detection in the factor VIII gene is complicated by the size and complexity of the gene—186 kb spanning 26 exons. The exons vary in size from 69 bp to 3106 bp and the introns from 207 bp to 32.4 kb (1). The first mutations to be identified in the factor VIII gene involved mutations at Taq I restriction sites (an e ...

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Screening for DNA Heteroduplexes in the Factor VII Gene Using Ethylene Glycol Gel Electrophoresis of Solvent-Treated 32P-Labeled PCR Products

Sequencing exon by exon to identify mutations is both laborious and time consuming. Screening techniques have been sought to identify mutations without the need for this. Rapid detection of single base changes have focused on identification of the heteroduplex formed when wild-type and ...

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Screening for Mutations in the Human Antithrombin Gene by Hydrolink D-5000 and MDE Gel Electrophoresis

The polymerase chain reaction (PCR) provides a rapid method for generating a large amount of a defined region of DNA without recourse to cloning. However, direct sequencing of this amplified material is tedious, time-consuming, and frequently generates large amounts of normal sequence d ...

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