遗传学实验技术

Advances in our understanding of the protozoan parasite Leishmania have been facilitated by the development of molecular and genetic tools. One powerful approach for gene identification and analysis is transposon mutagenesis. This can be performed directly in vivo, but often it is more ...

In 1980, Gordon et al. (1) showed that DNA injected into the pronuclei of single-cell embryos could be incorporated, expressed, and transmitted to the offspring of transgenic mice. Since then, pronuclear injection has become a widely used and invaluable tool for the study of mammalian gene funct ...

The generation of germline transformants in Drosophila melanogaster has relied on the utilization of transposable elements to effect the chromosomal integration of injected DNA (1,2). The success of this approach has depended largely on our understanding of the biology of P elements a ...

Replication-defective adenoviruses (Ads), in which the E1 A and E1B genes essential for replication are deleted, have several advantages as the vectors for introducing foreign genes into host cells. They are able to infect a large range of host cells and to transfer genes into nonproliferati ...

To provide fertilized eggs for microinjection, a production colony needs to be established. This should be carefully planned in order to provide enough material for your requirements. There are several items for consideration in this regard, detailed in the following sections.

Retrovirus vectors have been used for a variety of applications requiring gene transfer, including the production of transgenic animals. In all such cases, the transgene is delivered through an infectious particle and as part of a retrovirus genome. In almost all cases, the retrovirus geno ...

Embryonic stem (ES) cells are derived directly from those undifferentiated progenitor cells of early mouse embryos that have the developmental potential to subsequently form all the tissues of the fetus itself. With appropriate culture conditions, these embryonic cells can be main ...

Embryonic stem (ES) cells are undifferentiated cells derived from early mouse embryos, which under appropriate culture conditions proliferate continuously in vitro. ES cells have been demonstrated to be pluripotent in vivo from their capacity to form teratocarcinomas and germl ...

Embryonic stem (ES) cell technology has clearly established itself as a powerful technique for the examination of gene function in vivo. The vast majority of gene-targeting experiments to date have been designed simply to inactivate the function of the gene of interest by the targeted inser ...

Cryopreservation can help minimize waste and thereby increase the efficiency with which transgenic mouse colonies can be used (1,2). The main applications for cryopreservation are firstly the longterm storage of transgenic mouse lines and secondly the shortterm storage of embryos ...

A major problem for research groups breeding inbred, mutant, and transgenic mouse lines is that many are poor breeders, or do not breed at all. This problem is often difficult to alleviate. Sperm banking can be used for mice (1–;3), but in some instances it is also important to bank the female genome (4). Supero ...

The first transgenic experiment was carried out more than 15 yr ago, and transgenesis is now a routine technique for the study of gene and cell function, and the development of animal models of disease. The mouse remains the species of choice for the vast majority of transgenic applications, reflect ...

The first transgenic livestock were reported in 1985 (1). The techniques for producing these animals used pronuclear injection, which had been established previously in the mouse (2). This technique involves the direct introduction of a few hundred copies of a DNA construct into one of the two p ...

Recent progress in the production of mammals by nuclear transfer using donor nuclei from cultured cell populations has provided a novel route for genetic manipulation. The technique of nuclear transfer allows the production of offspring by the reconstruction of an embryo. Genetic mate ...

The first step in the analysis of potentially transgenic mice is the characterizaton of their altered genotype. A strategy must be devised that allows easy and reliable discrimination between wild-type animals and those carrying additional transgenes or introduced targeted mutat ...

Vertebrate genomes are globally heavily methylated at the sequence CpG, with the exception of short patches of GC-rich DNA of between 1–2 kb in size that are free of methylation, and these are known as CpG islands (see refs. 1 and 2 for reviews). In addition to distinctive DNA characteristics, CpG islands a ...

DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (review refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiating c ...

The reaction catalyzing direct demethylation of DNA involves the removal of a methyl group residue from the 5′ position on cytosine; the products of the reaction are nonmethylated cytosine in the dinucleotide CpG and methanol (1). The study of the proteins involved in demethylation requires ...

Purification of an enzymatic activity requires a simple and relatively expeditious assay of its activity. However, the nature of the demethylase reaction has been elusive for decades. Although a large body of evidence supported the hypothesis that active demethylation takes place du ...

Nearest-neighbor analysis can be used to identify the 3′ nearest neighbors of 5mC residues in DNA (1,2). It can also be used to measure the level of methylation of a specific methylated dinucleotide in DNA. Typically, in the case of mammalian DNA, this means quantifying the degree of methylation at CpG di ...