Jonathan D Gilthorpe and Peter W J Rigby 1 Introduction

The development of transgenic technology during recent years has allowed researchers to probe much more deeply than was previously possible into the molecular mechanisms influencing embryonic development. Transgenic procedures allow the transfer of a cloned gene into a host genome or the mutation of specific genomic sequences via targeting in embryonic stem (ES) cells (see Chapter 7). Most commonly, transgenic gene transfer experiments have been used to study the phenotypic effects caused by the misexpression or overexpression of a transgene (1-3), or to investigate the transcriptional mechanisms underlying developmental and tissue-specific gene regulation (4-6). The use of reporter genes in this latter application will be dealt with in this chapter.

Prior to the advent of gene transfer methods, research into the developmental regulation of gene expression was restricted to the use of indirect embryo-logical and in vitro techniques. However, transgenesis employing reporter genes has provided a model assay to identify cis-acting DNA sequences that can influence particular aspects of gene expression during development in vivo. Providing that the transgene integrates into the germline of the founder transgenic animal, it will be present in every cell of the F1 generation, and may be passed on to subsequent generations. This enables the expression of a gene to be studied throughout development and in all cell types, which is not possible using cell-culture techniques. The system employs a transgene consisting of genomic sequences linked to a reporter gene, the product of which can be easily detected. The expression of the wild-type construct and of truncated or mutated forms can be monitored by histochemical analysis and compared, thus

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

allowing specific regulatory sequences to be defined. In the final analysis, individual transcription factor binding sites can be identified enabling the characterization of the cognate trans-acting factors. The most suitable reporters for this purpose are genes encoding enzymes, the activity of which can be detected with a chromogenic substrate enabling expression patterns to be directly visualized. Two such reporter genes are currently available, the Escherichia coli lacZ gene encoding P-galactosidase, and the human gene encoding placental alkaline phosphatase (PALP-I).

Three basic methods are available for the production of transgenic mice carrying reporter genes. The manipulation of ES cells and retroviral infection of embryos are useful for certain types of experiments (7). The most commonly used method, however, is the microinjection of DNA into the pronuclei of single-cell embryos (8), and this is best suited to studies of gene regulation. This process begins with the construction of a suitable transgenic reporter construct, which is then purified and microinjected into fertilized eggs. Microinjection is a time-consuming procedure requiring a high degree of skill. An experienced operator can microinject and transfer between 150 and 300 fertilized eggs/d. Approximately 30-60% of these will develop to give viable embryos, of which 10-20% will usually carry the transgene and a proportion will express it in a construct-dependent manner. Generally this translates to 1-3 d of injection/construct to give a reliable number of data points (ideally six or more expressing mice or embryos). In addition, an expensive microinjection setup is required as is a well-organized animal facility to provide and maintain the mice.

This chapter covers the design, construction, and purification of a reporter gene for microinjection and the subsequent analysis. Staining protocols for P-galactosidase and alkaline phosphatase activities are included. Details of transgenic mouse production have not been incorporated, and readers should refer to earlier chapters in this book and to the "transgenic bible" of Hogan et al. (7).

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