遗传学实验技术

The easiest and cheapest way to analyze the phenotype of most knockout mice is to do a comprehensive necropsy and histopathologic examination of slides of all tissues. Once any lesion is found in a knockout mouse a vast contemporary and traditional literature can be searched for occurrences of s ...

The history of mouse genetics, which involves the study of strain-dependent phenotype variability, makes it clear that the genetic background onto which a gene-targeted allele is placed can cause considerable variation in genetically engineered mouse (GEM) phenotype. This variat ...

Since the mouse has become the most profound model system to investigate the genetics and pathogenetics of human diseases, a huge number of new mutant mouse strains has been generated and still a lot effort is being done to increase the number of suitable mouse models. In nearly all animal facilities t ...

Lentiviral vectors efficiently transfer genes into a broad spectrum of cells and tissues, including non-dividing cells and stem cells. Lentiviruses integrate their viral genome into the host chromosome, which is the basis for virus latency as well as stable transgene expression. A rath ...

Understanding the functional landscape of the mammalian genome is the next big challenge of biomedical research. The completion of the first phases of the mouse and human genome projects, and expression analyses using microarray hybridization, generate critically important que ...

The Cre/lox site-specific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for ...

Generation of genetically modified mice is one of the primary methods for understanding gene function. In particular, approaches that allow for restricting the effects of a mutation to defined cell-types are fundamental for understanding the roles of genes in specific cells or tissues. T ...

With the completion of the human and mouse genome sequences and the development of high-throughput knockout mouse technologies, there is now a need for equally high-throughput methods for the production of mice for phenotypic studies. In response to this challenge, we recently developed a ...

During the past 40 years, mouse chimeras have served as invaluable tools for studying not only genetics but also embryonic development, and the path from undifferentiated cell populations to fully committed functional cell types. This chapter gives a description of the early events of cell ...

Genetically modified mice by means of homologous recombination in embryonic stem (ES) cells are generated by injection of manipulated ES cells into recipient blastocysts. The injected blastocysts, following reintroduction into recipient foster mice, will produce chimeric mi ...

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all o ...

Pluripotent embryonic stem (ES) cells are characterized by their almost unlimited potential to self-renew and to differentiate into virtually any cell type of the organism. Here we describe basic protocols for the in vitro differentiation of mouse ES cells into cells of the cardiac, neuron ...

A method is described to establish mouse embryonic stem cell (ESC) lines from hybrid and inbred strains of mice. Attention is paid not only to the methodology for isolation and culture but also to the validation of freshly derived lines, in order to be maintained for prolonged time without signific ...

Unraveling the function of the mammalian genome relies heavily on analyses of the laboratory mouse. Because of its powerful genetics and available technologies to manipulate the genome, plus its developmental and physiological similarities to humans, it has become a goal to generate m ...

RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a faster alternative to conventional knocko ...

Oligonucleotide-mediated gene targeting is an attractive alternative to current procedures to subtly modify the genome of mouse embryonic stem (ES) cells. However, oligonucleotide-directed substitution, insertion or deletion of a single or a few nucleotides was hampered by DNA m ...

Chromosomal rearrangements, such as deletions, duplications, inversions and translocations, occur frequently in humans and can be disease-associated or phenotypically neutral. To understand the genetic consequences of such genomic changes, these mutations need to be mode ...

Gene trapping can be used to introduce insertional mutations into the genome of mouse embryonic stem cells (ESCs). The method has been adapted for high-throughput use, in an effort to inactivate all genes in the mouse genome. Gene trapping is performed with vectors that simultaneously inacti ...

In this chapter we give an overview of mutagenesis methods in the mouse as they evolved over the last two decades, an outlook of ongoing and future developments and advice for choosing a mutagenesis strategy. Where appropriate, reference is given to relevant chapters of this book, key original art ...

Delivering exogenous DNA or genes directly to the lung airways offers a unique and appealing opportunity for specifically targeting gene expression to airway and alveolar epithelium. A large body of literature and experience supports the feasibility of this approach. However, airw ...