2.1. Application of Transgenic Mouse Technology
Transgenic mice are widely used in biomedical research, and numerous applications have been developed, including those used in hypoxic pulmonary hypertension (5, 6, 19, 29). There are two common uses of transgenic mice in biomedical research. The first is to study the phenotypic effects of transgene expression in the intact animal. In this case, previously defined promoters are often used to control the expression of the transgene in the desired tissue. The second is to study the control of gene expression in the intact animal. For this purpose, potential control sequence elements (promoter regions) are used to define the specific patterns of the transgene expression in various tissues.
For applications involving the phenotypic effects of transgene expression, transgenic mice expressing the given genes, in general, represent gain-of-function mutations, whereas loss-of-function mutations in many cases can be obtained solely by gene targeting (see below). However, dominant negative mutations can also be obtained through expression of some mutant forms of genes (10-13). In addition, the expression oftransgenes encoding antisense RNA or short interfering RNA successfully inhibited the expression of endogenous genes in transgenic mice (8, 24).
Most cloned genes introduced into the mouse germ line have shown appropriate tissue-specific and stage-specific patterns ofexpression despite their integration into apparently random sites in the host genome. Thus, for studies of the control of gene expression, transgenic mice have provided the definitive experimental assay to define the cis-acting DNA sequences that control specific patterns of transcription in vivo (2, 7, 25).
The procedure for generating a conventional transgenic mouse line includes preparing DNA constructs, setting up the mouse colony, microinjecting DNA constructs into the pronuclei of fertilized eggs, characterizing transgenic "founder" mice, and generating transgenic lines from these founder mice. The quality of the DNA constructs is critical for efficient generation of transgenic founder mice. High purity ofthe DNA, avoiding the use ofethidium bromide for DNA staining, removing all vector sequences from the cloned genes, and using relative short DNA constructs (<70 Kb) usually increase the rate of success (4060%) of generating founder mice.
The most extensively and successfully used method of gene transfer is microinjection of DNA directly into the pronuclei of fertilized mouse eggs. Infection of embryos with retroviral vectors has also been used. The microinjection method results in the stable chromosomal integration of the foreign DNA in 10-60% of the resulting mice. In most cases, the integration appears to occur at the one-cell stage of the embryos; as a result, the foreign DNA is present in every cell of the transgenic mouse, including all primordial germ cells. In 15-25% of cases, foreign DNA is integrated at a later stage, resulting in mice that are mosaic for the presence of foreign DNA.
2.3. Generation of Conditional (Inducible) Transgenic Mice
Sometimes, it is desirable to have transgenes that will be silent until specifically activated by an experimental manipulation, such as the administration of a drug. In early studies, the metallothionein promoter was frequently used to generate inducible transgenic mice (26). The metallothionein promoter drives transgene expression at low basal levels in many tissues;
however, feeding the transgenic mice with water containing Zinc can increase the transgene expression up to 100-fold in many tissue, including liver, kidney, and intestine (26). However, this system lacks tissue specificity.
Recently, the tei-operon (Tet-O) system has been used widely to generate conditional (inducible) transgenic mice in a tissue-specific manner (22, 23). In this system, two transgenic mouse lines must be generated: one with the desired transgene under the control of a Tet-O promoter containing a tetracycline-responsive element (TRE) and the other expressing a tetracycline-controlled transactivator (tTA) under the control of a tissue-specific promoter (Fig. 1). Crossing these two lines will generate transgenic mice carrying both transgenes. When the doubly transgenic mice are fed a diet containing tetracycline, the tetracycline binds to tTA, blocking the binding ofthe tTA to the TRE of the TetO promoter and turning off the transgene expression (Fig. 1). When the tetracycline is withdrawn, tTA is released and binds to the TRE of the Tet-O promoter, turning on the transgene expression (Fig. 1).
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