遗传学实验技术

Since the technique of introducing a targeted mutation (‘gene targeting’) into the mouse genome was published almost 20 years ago (Cell 51:503–512, 1987), the number of mouse mutants (mouse models) is increasing, especially after the advent of the full mouse genomic sequence in 2002 and the human g ...

This chapter describes the tools and the experimental route of targeted manipulation by aggregation in the mouse using targeted embryonic stem cells (ES cells). Instead of injecting ES cells into the blastocoel of a diploid blastocyst-stage embryo (3.5 dpc) ES cells can be brought together w ...

Site-specific recombination systems are widespread and popular tools for all scientists interested in manipulating the mouse genome. In this chapter, we focus on the use of site-specific recombinases (SSR) to unravel the function of genes of the mouse. In the first part, we review the most comm ...

The previous two chapters have described the generation of chimeric embryos by blastocyst microinjection and morula aggregation. This chapter describes the reimplantation of these embryos into pseudopregnant female recipient mice (foster mice) in order for the embryos to devel ...

With the development of the Cre-LoxP system, conditional gene targeting has rapidly become a powerful technology that facilitates the study of gene function. This advanced technique circumvents three major concerns sometimes levelled against conventional transgenic and gene ...

The complexity of mammalian biology, in combination with the ability to manipulate the embryonic genome in mice, has provided specific challenges in terms of mouse breeding, analysis, and husbandry. From the initial planning stage of a project involving the generation of novel, genetica ...

Determining gene function by using transgenesis in the mouse has been immensely popular amongst researchers since the introduction of the techniques in the 1980s. However, we are still a long way from knowing the function of the majority of the genes in the genome. It is becoming increasingly acc ...

The mouse is now firmly established as the model organism of choice for scientists studying mammalian biology and human disease. Consequently, large collections of novel genetically altered mouse lines have been deposited in secure archives around the world. If these resources are to be of ...

The mouse is now firmly established as the model organism of choice for scientists studying mammalian biology and human disease. Consequently, a plethora of novel, genetically altered (GA) mouse lines have been created. In addition, the output from the large scale mutagenesis programmes c ...

Since the early 1980s, when the first transgenic mice were generated, thousands of genetically modified mouse lines have been created. Early on, Jaenisch established proof of principle, showing that viral integration into the mouse genome and germline transmission of those exogenous ...

Pronuclear injection is a mode of DNA delivery for the production of transgenic animals. We present protocols for the preparation of DNA for microinjection from small or large plasmids and from BACs. We describe the production of one-cell mouse embryos, unfertile males, and pseudo-pregnant ...

Mouse transgenesis by pronuclear injection generates random integration events resulting in variable overexpression efficiency. This chapter will outline current strategies for improved transgene design (using plasmids or bacterial artificial chromosomes) and del ...

Gene targeting in mice has been used extensively to elucidate gene function in vivo. However, for gene targeting to be successful, the targeting vector must be carefully designed. This chapter addresses the rationale behind designing targeting vectors, detailing the essential compo ...

Alteration in epigenetic regulation of gene expression is a common event in human cancer and developmental disease. CpG island hypermethylation and consequent gene silencing is observed for many genes involved in a diverse range of functions and pathways that become deregulated in the ...

Central to systems biology are genome-wide technologies and high-throughput experimental approaches. Completion of the sequencing of the human genome as well as those of a number of other higher eukaryotes now allows for the first time the mapping of all of the cis-regulatory regions of genes ...

Genome-wide ChIP-chip assays of protein–DNA interactions yield large volumes of data requiring effective statistical analysis to obtain reliable results. Successful analysis methods need to be tailored to platform specific characteristics such as probe density, genome cov ...

Historically, the simplest method to robustly identify active gene regulatory elements has been enzymatic digestion of nuclear DNA by nucleases such as DNaseI. Regions of extreme chromatin accessibility to DNaseI, commonly known as DNaseI hypersensitive sites, have been repeate ...

Over the past decade, DNA microarrays have proven to be a powerful tool in biological research for the molecular surveillance of cells and tissues. The expansive utility of DNA microarrays owes its nascence to the development of a multitude of microarray platforms that enable the systematic a ...

Alterations in genomic DNA are a key feature of many constitutional disorders and cancer. The discovery of the underlying regions of gene dosage has thus been essential in dissecting complex disease phenotypes and identifying targets for therapeutic intervention and diagnostic te ...

Recent advances in DNA microarray technology have enabled researchers to comprehensively characterize the complex genomes of higher eukaryotic organisms at an unprecedented level of detail. Array-based comparative genomic hybridization (Array-CGH) has been widely used f ...