遗传学实验技术

Over the past decade, a number of techniques have been developed that allow the introduction of defined, cloned DNA sequences into animal germ lines. Once inserted, these sequences, now called transgenes, are stably passed on from generation to generation. In other words, the transgene becom ...

The production of transgenic sheep has proven difficult compared to the mouse and lower animals. The work load is far greater and the rates of success far less by most criteria. However, the benefits to human and animal health and agricultural productivity are potentially enormous (Ward and Nan ...

The following protocol is for freezing rat blastocysts by direct plunging into liquid nitrogen (1). Although this protocol is designed for freezing the embryos in polypropylene cryotubes, it could readily be adapted to 0.5-mL straws. For a general discussion of cryopreservation, the rea ...

The harvest of fertilized one-cell eggs of rats is very similar to mice (see Chapter 17). However, in the case of rats, collagenase is used instead of hyaluronidase for the removal of cumulus mass from around the eggs.

A transgenic animal represents an enormous investment in time and money. Animals can be destroyed through disease, fire, malfuncnons in the control of the environment, negligence, sabotage, or accidental disposal. Researchers can protect valuable transgenic lines from accrdent ...

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 (1,2) and ge ...

Some transgenic animals may prove to be infertile through an inability to mate or rear a litter. It will therefore be necessary to perform an in vitro fertilization (IVF) procedure (1) in order to continue the line. The technique involves superovulating females and fertilizing the eggs with sperm ...

Following superovulation and mating (see Chapter 13), the immature F1 females are killed, and the fertilized F2 eggs are dissected out from the upper part of the oviduct or ampulla. The procedure for egg collection (1) is described in the present chapter. Eggs may be recovered several hours before th ...

Two types of culture media (1) are required for the in vitro manipulations involved in the process of making transgenic mice. (1) M16: This is used for maintenance of eggs in microdrop cultures (see Chapter 17) in a 37�C incubator gassed with 5% CO2. Since this medium is buffered with bicarbonate alone and the ...

Vasectomized males are needed to engender pseudopregnancy in mature female mice. Such pseudopregnant females act as hormonally and physiologically competent recipients for microinjected fertilized one-cell eggs (see Chapter 20). Vasectomized males retain their sexual po ...

Dual-color fluorescent cells, with one color in the nucleus and the other in the cytoplasm, enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) is expressed in the cytoplasm of can ...

The unraveling of the complex dynamic networks that underlie cellular (and, by extension, tissue, organ, and organism) function requires sophisticated mathematical models and, in order to test those models, rich data sets. In addition, even in clonal populations of cells, there is a wide range ...

The unique spectral properties and versatility of autofluorescent proteins have facilitated their widespread use in flow cytometric applications. The ability to analyze heterologous fluorescent protein expression conveniently and noninvasively by individually in ...

Using the vital marker GFP and its spectral variants, it is possible to visualize multiple proteins in individual cells and thereby monitor embryonic development on a cellular and molecular level. In the following chapter we describe how to prepare Drosophila embryos or larvae for live imag ...

Through the use of exogenous labels, such as antibodies and synthetic fluorophores, experimenters have been able to readily observe the localization of proteins and organelles within a cell by fluorescence microscopy. The discovery and application of fluorescent proteins spann ...

This chapter describes two episcopic imaging methods, episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM). These allow analysis of molecular signals in a wide variety of biological samples such as tissues or embryos, in their precise ana ...

We have shown that to overcome the low levels of expression from gene transfer vector-mediated β-galactosidase expression in lung, it is essential to replace the cytoplasmic β-galactosidase gene with a nuclear targeted β-galactosidase gene. We found that lung should be sectioned and fix ...

Green fluorescent protein (GFP) and its variants, small, highly soluble proteins, are routinely used as reporters for patterns of gene expression and the origin of cells in transplantation experiments. When not linked as fusion proteins to other polypeptides, they distribute rapidly in ...

Bioluminescence imaging has become a very popular tool for noninvasive monitoring of fundamental biological and molecular processes in small living subjects. Luciferases are light-emitting enzymes that can generate light (known as bioluminescence) after reacting with spe ...

This chapter provides information on β-galactosidase staining of whole mouse embryos, organs, tissue sections, and cultured cells, as well as double staining with horseradish peroxidase and use as a tool for genotyping. Using these protocols, localization of β-galactosidase can be vi ...