Analysis of Reporter Gene Expression

Owing to the large amount of work required for the production and analysis of transgenic mice, it is important to employ careful planning in the design and execution of experiments. The rate-limiting step in the process is the production of transgenics, so any information that can reduce the number of constructs required can greatly speed up the operation (see Notes 33 and 34).

Once a baseline reporter has been defined, it is extremely important to establish clearly its characteristics of expression throughout various developmental stages. This will enable subsequent comparison with mutant constructs and aid the definition of specific regulatory elements. Because of the enormous variety of results that can be obtained from such work, it is impossible to advise a standard schedule for priorities. It is advisable though to be very thorough with the analysis and to review continually the situation as new data arrive (see Note 35).

Once regulatory elements have been localized to relatively small regions (e.g., a few hundred basepairs), it is often better to employ in vitro methods (e.g., electrophoretic mobility shift assays or footprinting) and sequence analysis for the identification of specific potential regulatory elements. This may be much more rapid than continued mutational transgenic analysis. When individual transcription factor binding sites have been identified, the effect of mutations in them can be further tested in the transgenic context.

3.6.1. Transient vs Line Analysis

Many benefits can be drawn from directly analyzing founder transgenic embryos (transients) rather than breeding them through to the next generation and producing a transgenic line. The disadvantage is that each embryo repre sents only a snapshot of a particular expression pattern at a particular developmental time-point. However, it does allow the rapid analysis of a large number of transgenic constructs without the need for a time-, space- and labor-intensive breeding program. Transient analysis is therefore recommended for an initial assessment of a large number of constructs and line analysis for more specific, in-depth analyses.

3.6.2. Position Effects and Ectopic Expression

The position of integration of a transgene in the host genome is considered to be random, and it can greatly affect both the levels and pattern of its expression (termed position effects). A certain proportion of transgenic animals will not express the reporter gene either as a result of rearrangements during integration or because integration has occurred in a region of inactive chromatin. Position effects are often more evident with smaller or mutated transgenic constructs where the reporter is presumably more exposed to interference from surrounding regulatory elements. It should also be noted that there are reported instances where P-galactosidase activity does not reliably report levels of transcription (see Note 36).

Two forms of ectopic expression from a transgenic reporter are possible: Inconsistent ectopic expression owing to position effects is discernible by the comparison of a number of transgenic animals carrying the same construct. Consistent ectopic expression owing to regulatory interactions within the reporter gene per se is less clear-cut, since they may only be apparent in a proportion of the analyzed samples (if dependent on expression levels, and so forth). Another source of consistent, but apparently ectopic expression is an unavoidable drawback of reporter genes, and is a function of the reporter gene products (mRNA and protein) and their stability within the cellular environment (see Note 37).

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