基因表达差异显示实验

Although a method exists for freezing sheep embryos directly into liquid nitrogen without a programmable freezing machine (1), a more traditional slow cooling method is described here. Either dimethyl sulfoxide (DMSO) or ethylene glycol can be used as a cryoprotectant in this slow cooling ...

Microinjected eggs are normally incubated overnight in Ml6 microdrop culture at 37�C in CO2 until 1400 h. By this time the eggs will have developed as far as the two-cell stage. The eggs are then implanted into surrogate mothers. As the membrane covering the oviduct of the rat is much tougher than the mouse, we ...

Mature male rats that are sexually active are vasectomized and used to produce pseudopregnancy in sexually mature female rats. These pseudopregnant female rats are used as surrogate mothers. Microinjected fertilized one-cell eggs are implanted into the oviduct of such recipients. As ...

For the production of transgenic rats, matings are carried out under controlled conditions in order to obtain a supply of one-cell fertilized eggs, and to obtain pseudopregnancy in females, which will act as surrogate mothers for the microinjected eggs.

Until recently, the mouse has been the animal of choice for transgenie studies. This is because the scientists who pioneered transgenic technologies emerged from the fields of mouse genetics and embryology. The mouse will continue to be the most convenient mammalian model for asking devel ...

This chapter seeks to outline the strategies being employed to exploit transgenic mice as a tool in the study of cancer. Rather than catalog the many different oncogene and oncogene-promoter combinations that have been introduced into transgenic mice to elicit tumors, I will concentrate on a ...

Embryonic stem (ES) cells are derived directly from those progenitor cells of early mouse embryos that subsequently form all of the tissues of the fetus itself and that under appropriate culture conditions, can be maintained continuously in an undifferentiated state in vitro (1–3; see Note 1). ...

There is great variation in the survival rate of microinjected eggs introduced into pseudopregnant recipient female mice. If the survival rate is low, then the few eggs that do develop tend to be “overnourished” and grow into larger fetuses compared to the embryos of a normal-sized litter. Under s ...

Following the microinjection procedure (see Chapters 18 and 19), the eggs must be transferred to pseudopregnant (0.5 d pc) recipient mothers. Embryos from the one-cell through the morula stage (0.5–2.5 d pc) are transferred into the ampullae by a procedure called an oviduct transfer (1), describ ...

In recent years, a number of automatic microinjection systems have appeared on the market. These systems replace the simple manual syringe system for forcing the DNA solution out of the microinjection pipet and into the pronucleus of a fertilized one-cell egg. The advantages of such automatic ...

Central to the process of making transgenic mice is the physical introduction of cloned DNA fragments into fertilized one-cell mouse eggs. First described 10 years ago by a number of investigators (1–5), microinjection remains the most popular and successful of the methods currently avai ...

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 ...