purposes related to mechanisms of developmental toxicity and polymorphic variation in susceptibility of toxicants. The incisive use of cultured cells has been enhanced greatly by molecular methods in which genes of choice (from any organism including humans) can be introduced into such cells, for example, to express variant forms of DMEs and to investigate the relation of function to allelic variation.
Human allelic variants code for gene products different from those encoded by the wild-type allele, and it is important to characterize and, if possible, quantify any functional alterations. The fundamental techniques available for studying gene expression include (1) expression in vitro where DNA from the allelic variant or wild-type allele is transcribed to the mRNA and then translated into a functional, active protein in a cell-free extract; (2) high-level expression in cells from which proteins can be purified with relative ease; and (3) expression in eukaryotic cell culture in which the cell or the cell’s DNA has been modified (i.e., genetically engineered). These techniques rely on the gene (or at least the cDNA-encoded “coding region” responsible for generating the amino acid sequence of the protein). The cDNA encoding the allele being studied must first be cloned into an appropriate plasmid vector that includes regulatory sequences that drive and terminate transcription and a selectable marker gene. As outlined below, each of the aforementioned expression strategies is used by the investigator to meet specific needs. In general, these in vitro and cell-culture systems are very useful, because they are relatively quick, efficient, inexpensive, and use simple technologies when compared with the development of mouse lines or other animal-model systems.
There is a major shortcoming, however, in the in vitro and cell-culture expression systems. Single-nucleotide polymorphisms (SNPs) outside the amino-acid-determining region of the gene (e.g., splice junctions; promoter sequence; 5′ and 3′ untranslated regions; and enhancers upstream, downstream, and inside the gene) can have striking effects on expression of the gene under study, and the effects would generally not be realized, characterized, or quantitated by the in vitro and cell-culture expression systems (Nebert 1999).
There are both reticulocyte lysate and wheat-germ lysate combined transcription and translation kits now commercially available to assess the interaction of the protein under study with another purified protein. To use these systems, a plasmid containing the cDNA of interest and a radiolabeled amino acid are added to the lysate. Although the protein of interest is not expressed at high levels relative to the total amount of proteins in the lysate, it is the only radiolabeled protein in the reaction mixture.