遗传学实验技术

The human respiratory pathogen Haemophilus influenzae, a Gram-negative bacterium, is the first freeliving organism to have its complete genome sequenced, providing the opportunity to apply genomic-scale approaches to study gene function. This chapter provides an overview of a hi ...

We present a conceptual review of transposition-based strategies for determining gene essentiality on a one-by-one basis in bacteria. Many of the techniques are described in greater detail in individual chapters of this volume. The second section of this chapter deals with transposit ...

A method is described for generating conditional lethal mutations in essential genes in Escherichia coli. In this procedure, amber stop codons are introduced as “tagalong” mutations in the flanking DNA of a downstream antibiotic-resistance marker by lambda Red recombination. The ma ...

This chapter provides methods and insights into the use of antisense RNA as a molecular genetic tool. Posttranscriptional inhibition of specific gene expression can be achieved by antisense RNA fragments under control of a conditional promoter. Effective titration of gene expressi ...

Genome-scale metabolic models of organisms can be reconstructed using annotated genome sequence information, well-curated databases, and primary research literature. The metabolic reaction stoichiometry and other physicochemical factors are incorporated into the m ...

Novel genetic tools and mutagenesis strategies based on the Sleeping Beauty (SB) transposable element are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of its inherent capacity to insert into DNA, the SB ...

Intact chromosomes as well as chromosome fragments can be vehicled into various recipient cells without perturbing their ability to segregate as free elements; chromosome transfer can be performed both in cultured cells and in living animals. The method of choice to shuttle single chrom ...

Artificially modified chromosome vectors are non-integrating gene delivery platforms that can shuttle very large DNA fragments in various recipient cells: theoretically, no size limit exists for the chromosome segments that an engineered minichromosome can accommodate. Th ...

Extrachromosomal gene expression vectors that contain native genomic gene expression elements have numerous advantages over traditional integrating mini-gene vectors. In this protocol chapter we describe our work using episomal vectors where expression of a cDNA is control ...

Extrachromosomal, or episomal, vectors offer a number of advantages for therapeutic and scientific applications compared to integrating vectors. Extrachromosomal vectors persist in the nucleus without the requirement to integrate into the host genome, hence avoiding the rec ...

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly, and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with pl ...

Modifying multipotent, self-renewing human stem cells with mammalian artificial chromosomes (MACs), present a promising clinical strategy for numerous diseases, especially ex vivo cell therapies that can benefit from constitutive or overexpression of therapeutic gene(s). ...

Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are l ...

Horizontal gene transfer or simply transgenic technology has evolved much since 1980. Gene delivery strategies, systems, and equipments have become more and more precise and efficient. It has also been shown that even chromosomes can be used besides traditional plasmid and viral vectors ...

Gene therapy encounters important problems such as insertional mutagenesis caused by the integration of viral vectors. These problems could be circumvented by the use of mammalian artificial chromosomes (MACs) that are unique and high capacity gene delivery tools. MACs were delive ...

Mammalian satellite DNA-based artificial chromosomes (SATACs) are unique among the mammalian artificial chromosomes. These reproducibly generated de novo chromosomes are stably maintained in different species, readily purified from the host cell’s chromosomes and can be i ...

Among the many techniques of cloning new genes, one approach involves degenerate primers (1–7). The approach usually requires the following three steps: 1. Using degenerate primers to amplify part of the gene of interest by PCR: The degenerate pri

A crucial and often decisive test of a nuclear gene being involved in a given process is the complementation of mutants. Restoring the wild type phenotype by the wild type gene introduced into the mutant is a major piece of evidence for the function of this gene. We have developed a rapid and reliable method to ...

Although type I topoisomerases do not require a high-energy cofactor, type II topoisomerases require ATP in order to carry out their essential catalytic functions (1–4). ATP binding is necessary to close the protein clamp (5,6) and trigger DNA strand passage (7,8), whereas hydrolysis is neces ...

This chapter outlines the generation and application of human papillomavirus type 33 (HPV33) pseudovirions. The method describes (1) the construction of vaccinia viruses recombinant for the major and minor HPV capsid proteins, L1 and L2, respectively; (2) the transfection of Cos7 cells ...