遗传学实验技术

This chapter describes the manual typing of STR markers, detailing the electrophoretic techniques used to separate amplified products, and the relevant methods for their detection.

Mitochondrial DNA (mtDNA) typing is increasingly used for the forensic identification of human remains (1-9). This is especially true when only limited quantities of sample are present, such as when the sample has undergone extensive degradation and nuclear-typing methods are ineffe ...

For separation of alleles of PCR-dependent short tandem repeat (STR) polymorphisms, high resolution with the power to discriminate fragments differing by one base pair is often desired. Furthermore, sequencing reactions also require unambiguous one base-pair separation. For bo ...

The isolation of genomic deoxyribonucleic acid (DNA) is a crucial step in the process of DNA profiling. The success of all subsequent genetic-typing procedures depends on the availability of sufficient amounts of highly purified DNA from biological crime stains as well as from reference b ...

Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic stress due to drought and/or salinity. To genetically transform the available commercial oat cultivars, a genotype-independent and efficient regeneration system from shoot apical merist ...

Ever since the first developments in plant transformation technology using model plant species in the early 1980s, there has been a body of plant science research devoted to adapting these techniques to the transformation of crop plants. For some crop species progress was relatively rapid, ...

The low frequency and randomness of transgene integration into host cells, combined with the significant challenges of recovering whole plants from those rare events, makes the use of selectable marker genes routine in plant transformation experiments. For research applications ...

We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used meth ...

Reporter genes have been widely used in plant molecular biology, typically to discern patterns of gene expression, but also as markers of transformed cells during stable transformation procedures. The ideal marker gene would be expected to display characteristics such as ease and chea ...

The engineering of novel properties and functions into viral vectors for improved gene delivery remains a barrier to the development of efficient, customized gene delivery vehicles. Rational methods for designing improved viral vectors are often experimentally challenging and ...

Photochemical internalization (PCI) is a physico-chemical targeting method that enables light directed delivery of nucleic acids into cells. The technology is based on photosensitizers that localize in the membranes of endocytic vesicles. A light activation of the photosensit ...

The recent development of leukemia in gene therapy patients with X-linked severe combined immunodeficiency disease because of retroviral vector insertional mutagenesis has prompted reassessment of the genotoxic potential of integrating vector systems. In this chapter, va ...

Vectors used in gene therapy require an expression cassette. The expression cassette consists of three important components: promoter, therapeutic gene and polyadenylation signal. The promoter is essential to control expression of the therapeutic gene. A tissue-specific prom ...

Transduction of therapeutic transgenes using multiply attenuated viral vectors is considered an essential technology for gene therapy scenarios. While first-generation viral transduction systems were engineered for constitutive expression of a single therapeutic tr ...

Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expr ...

Sleeping Beauty (SB) is a transposon system genetically reconstructed from teleost fish that mediates chromosomal integration of DNA sequences by a cut-and-paste mechanism. SB has been shown to mediate transposition in a variety of cells and tissues, has been used for the generation of tra ...

The recent development of artificial endonucleases with tailored specificities has opened the door for a wide range of new applications, including the correction of mutated genes directly in the chromosome. This kind of gene therapy is based on homologous recombination, which can be sti ...

Drug-inducible systems allowing the control of transgene expression and knockdown in mammalian cells are invaluable tools for genetic research, and could also play important roles in translational research or gene therapy. We and others have developed a lentivector-based, condi ...

Retroviral vectors have been used as gene delivery vehicles for more than two decades and continue to be the best available tool for stable and efficient transfer of therapeutic genes into various cell types. Although most gene therapy preclinical studies presently use crude or concentra ...

Adenovirus vectors currently are being evaluated in gene delivery studies ranging from prophylactic vaccination to therapeutic gene therapy. The quantity of purified virus required for these studies necessitate that purification methods must shift from classical density g ...