Mammalian molecular geneticists frequently are envious of the precision and speed with which the developmental biology of lower eukaryotes can be dissected. Deriving developmental mutants in mice by the use of traditional genetics, in which phenotype is selected followed by the unraveling of genotype, is far more complex and time consuming than in yeast, Drosophila and Caenorhabditis eiegans. However, the advent of technology for directly manipulating the mouse genome promises to contribute an arsenal of techniques for'reverse'genetics, whereby novel genes may be constructed and introduced into the genome to reveal the resulting phenotype. One of the more exotic approaches for reverse genetics developed in recent years is the insertion of genes that lead to programmed cell death by the developmentally controlled expression of a toxin. The use of toxins to study normal biology is not a new idea. The eminent physiologist Claude Bemard first suggested the use of toxins as probes for normal physiology, suggesting that,'Poisons are veritable reagents of life, extremely delicate instruments with which to dissect the vital units'(Bemard 1865). Immunologists have derived and applied'immunotoxins,'toxic substances coupled to monoclonal antibodies, to induce toxic ablation of specific cells and to attempt immunotoxic therapy of malignant diseases (Vitetta and Uhr 1985). However, the genetically programmed production of a toxin, rather than its pharmacologic use, recently has been applied through the construction of toxigenes. Toxigenes are artificial genes that produce a gene product that is itself a toxin or that has the capacity to metabolize a nontoxic substance to a toxin, leading to an induced'suicide'of the host cell. The potential of toxigenes for specific cell ablation results from the exquisite temporal and spatial control of gene expression during differentiation and the ability of disembodied regulatory signals to confer this specificity on artificial genes. Most recent work applying toxigenes as reverse genetic tools in transgenic mice is based on the earlier work of Maxwell et al.(1986), who studied the expression of the gene encoding the toxin produced by Corynebacterium clipbtheriae, the causative agent in human diphtheria, in transfected tissue culture cells. Diphtheria toxin is the product of the ton gene, which is carried by lysogenic corynephages of pathogenic strains of C. ch'phtheriae. As a prototype toxin for genetic ablation, diphtheria toxin consists of a toxic A subunit (DT-A) associated through disulfide bonds with a B subunit that is recognized by a specific receptor. Binding of the B subunit to the cell-surface receptor initiates internalization of the associated A subunit, which then catalyzes the NAD-dependent ADP ribosylation of elongation factor-2, resulting in the inhibition of protein synthesis and cell death (see Fig. 1).

A second potentially useful toxin for toxigene experiments is ricin, a toxic lectin produced by the castor bean, Ricinus communis, which is used widely for the assembly of immunotoxins and immunotoxic therapy. The structure of ricin is analogous to that of diphtheria toxin, in that the B subunit is a specific galactosyl binding protein that adheres to the extemal cell surface of virtually all cells presenting galactose-containing carbohydrates. The hydrophobic segment of the toxic A subunit {RA) allows it to penetrate the lipid bilayer and undergo conformational changes that lead to its release into the cytoplasm. Once internalized, RA acts as a highly specific ribonuclease that catalyzes the inactivation of ribosomes by cleaving the adenosine from the A4~ ss of the 28S ribosomal RNA (Lamb et al. 1985). The ability of these free RA …