遗传学实验技术

Lentivirus vectors are expected to become excellent tools for gene transfer in gene therapy and functional genomics and for engineering transgenic animals. When fully developed, their use will cover a whole gamut of endeavors, from discovery to therapeutics. Because they integrate in ...

The recovery of vectors that are suitable for an in vivo gene delivery has been a recurrent theme in gene therapy research over the last decade. Several challenging hurdles need to be overcome to reach such a goal. First, is a need for methods that allow the preparation of vectors at high titers and in culture s ...

Lentiviral vectors combine two useful vector properties: permanent integration and transduction of nondividing cells. Replication-defective lentiviral vectors were originally derived entirely from the human lentivirus human immunodeficiency virus type 1 (HIV-1) (1, ...

Lentivirus vectors are promising tools for gene transfer (1,2). Retroviral vectors in general offer the unique advantage of stably integrating into the genome of the host cell, thus providing the basis for sustained gene expression. In contrast to the classical oncoretrovirus-derived ...

Direct in vivo gene transfer to the central nervous system (CNS) using recombinant lentiviral vectors (rLV) has emerged as a powerful technique to overexpress various genes of interest in different neuronal populations. This interest is exemplified by the increasing number of studies ...

The goal of correcting such genetic lung diseases as cystic fibrosis could be achieved by vector-mediated gene transfer to airway epithelia. The principle of using viral vectors to complement genetic defects is well documented in cell culture and in animal models. Moreover, strategies for ...

An artificial chromosome is a synthetic structure that carries three fundamental components for its long-term survival, replication, and segregation after cell division. These components are telomeres, one or more replication origins, and a centromere. The creation of such a molec ...

Methods relating to the positioning of a transgene next to a newly formed telomere in human (HeLa) cells and the subsequent analysis of the resulting clones are described. These include vector design, analysis of integration sites by Southern blotting, pharmacological relief of silenci ...

Quantitative epigenetics (QE) is a new area of research that combines some of the techniques developed for global quantitative trait loci (QTL) mapping analyses with epigenetic analyses. Quantitative traits such as height vary, not in a discrete or discontinuous fashion, but continuo ...

Poly(ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that has traditionally been thought to require discontinuous or “damaged” DNA (dcDNA) as a coenzyme, a preconception that has limited research mainly to its role in cell pathology, i.e., DNA repair and apoptosis. Recent evidence has sh ...

The surge of interest in DNA methylation during the last two decades has triggered an urgent need for an effective method to detect the methylation status of the cytosines in the genome. Bisulfite genomic sequencing is the most attractive choice so far for many laboratories. Various protocols h ...

The last few years have seen a growing interest in the study of DNA methylation because of its now acknowledged implication in cancer. The use of bisulfite to convert unmethylated cytosine to uracil, even as methylated cytosine remains unchanged, constitutes the basis for differentiating ...

This chapter describes a detailed protocol using single-nucleotide primer extension (SNuPE) for quantitative analysis of DNA methylation on specific CpG sites. The first step DNA sample to be studied is treated with sodium bisulfite, which converts selectively unmethylated cyto ...

Denaturing gradient gel electrophoresis (DGGE) is a technique that fractionates DNA molecules on the basis of their melting behavior and thereby permits the separation of DNA fragments with local variations in base composition. The separation of DNA fragments by DGGE is determined by the ...

The best studied epigenetic modification in mammals is the methylation of cytosine. During the development and progression of malignant neoplasia, a global hypomethylation is often accompanied by a locus-specific increase in methylation. Also, during normal development spec ...

The phenotypic effects of aberrant gene expression are indistinguishable, regardless of whether the underlying mutation is one of gene copy number (deletion or duplication) or modification of differentially methylated CpG sites occurring in critical regulatory sequences in g ...

The methylation-specific oligonucleotide (MSO) microarray is a high-throughput approach capable of detecting DNA methylation in genes across several CpG sites. Based on the bisulfite modification of DNA that converts unmethylated cytosines to uracil but leaves the 5′methylc ...

Methylation-specific polymerase chain reaction (PCR) in situ hybridization, using paraffin-embedded, formalin-fixed tissues or formalin-fixed cell preparations, allows one to determine which specific cells have silencing of a given gene owing to hypermethylation of the pr ...

Epigenetics encompasses heritable changes in DNA or its associated proteins except mutations in gene sequence. Many investigators in the field of epigenetics focus on histone modifications and DNA methylation, two molecular mechanisms that are often linked and interdependent. A ...

DNA methylation is one mechanism of epigenetic gene regulation and influences gene expression by recruiting methylcytosine-binding proteins and/or inducing changes in chromatin structure. In mammals, DNA methylation is mediated by at least four DNA methyltransferase (Dnmt) ...