Operations, such as vasectomy (see Chapter 15) and oviduct transfer (see Chapter 20), are performed on anesthetized animals. It is essential that the experimenter is familiar with this procedure and that the anesthetic has been tested: A lethal dose of anesthetic administered prior to an ovid ...

In the process of creating and analyzing transgenic mice, matings between male and female animals are required for the following reasons: 1. To produce fertilized one-cell eggs for microinjection. Natural matings between mature females (over 6–7 wk of age) and stud male

A specific and dedicated mouse colony is required if transgenic mice are to be efficiently produced. The structure of such a colony is described in the present chapter. Although the provision of these animal facilities will not present a problem to large research establishments, smaller lab ...

The purification of a DNA fragment for microinjection is extremely important. This chapter describes a rapid and efficient technique for isolating specific DNA fragments from agarose gels run in Tris-acetate buffer, and was first described by Vogelstein and Gillespie (1). Agarose blo ...

Three basic techniques have emerged for making transgenic mice: two of these, retroviral infection of preimplantation embryos and the manipulation of embryonal stem cells, have attributes which make them desirable in some experiments, but the third technique, microinjection, is by f ...

Our understanding of the molecular mechanisms that govern gene expression has been facilitated by the ability to introduce recombinant DNA molecules into heterologous cellular systems both in vitro and in vivo. One approach to defining DNA sequences important in the regulation of gene ...

Reporter genes have become powerful tools to study regulation of gene expression in eukaryotes. In particular, chimeric transcription units generated by the fusion of the appropriate DNA regulatory sequences to reporter genes have led to the identification of a great number of DNA contr ...

The development of dominant selection markers to identify eukaryotic cells that have undergone a gene transformation event has greatly facilitated molecular genetic studies in higher eukaryotic cells. Selection schemes based on resistance to antibiotic cytotoxicity (1,2) w ...

Recombinant plasmid constructs are frequently employed in transfection experiments. With the availability of a wide spectrum of specialized and versatile eucaryotic cloning/expression vectors, investigators have been given powerful tools to expedite the elucidation of ...

The usefulness of the purine and pyrimidine salvage pathways in the study of the mechanisms of mutation and in the selection of cell lines stably transformed by vectors expressing these genes is well documented. Unfortunately, many investigators are deterred from selecting new host stra ...

The Sinuclease protection procedure allows the precise definition of the beginning and end of gene transcripts as well as the position of intron/ exon boundaries in a gene. Alternatively, when these parameters are already known, the technique can be used to quantify transcript levels in a vari ...

In the analysis of gene expression, the steady-state level of RNA transcripts is one of the most convenient parameters used to monitor the activity of an endogenous or introduced gene in cell lines and tissues. A variety of methods, such as Sl hybridization (Chapter 21, this vol), RNase protection (C ...

Primer extension is a relatively quick and convenient means by which transcription from a gene transfected into tissue-culture cells can be monitored. The technique can be used to accurately determine the site of transcription initiation or to quantify the amount of cap-site-specific m ...

The level of expression of a given gene in a particular cell type is reflected by the concentration of the resultant messenger RNA. This is subject to regulation during a number of processes, including synthesis, processing, export from nucleus to cytoplasm, and degradation. Clearly, the rate of ...

Antibodies, in general, provide the most sensitive and specific methods for detecting the protein products of genes. Immunoperoxidase techniques described here detect 103-105 molecules/cell (depending on whether the protein is dispersed within the cell or concentrated at high d ...

Multiplex polymerase chain reaction (PCR) refers to the simultaneous amplification of multiple regions of deoxyribonucleic acid (DNA) using PCR. Commercial short tandem repeat (STR) assays that can coamplify as many as 16 different loci have become widely used in forensic DNA typing. T ...

Because of their unique transmission properties and male specificity, markers located on the nonrecombining region of the Y chromosome (NRY) have become an important tool in forensic investigation. In the past few years, more than 50 polymorphic Y chromosome-specific short tandem rep ...

In this chapter we review and compare the online single nucleotide polymorphism databases that are now available as research tools. We give an outline of the search strategies that can be used to ensure the most appropriate loci for forensic applications are chosen.

Here, a single nucleotide polymorphism typing methodology is described based on polymerase chain reaction monitoring, in real time, of fluorescently labeled amplified products using the LightCycler. The main advantages of the system are the time required for the analysis (about 20 mi ...

Single nucleotide polymorphisms (SNPs) are emerging as new markers of interest to the forensic community because of their abundance in the human genome, their low mutation rate, the opportunity they present of analyzing smaller fragments of deoxyribonucleic acid (DNA) than with short t ...