usually fewer than 5 exons and introns can often be transfected transiently or stably into cells in culture as the complete gene. Dozens of promoters have been studied over the past 15 years. They include the Drosophila heat-shock promoter (Hsp), the mouse mammary tumor virus long-terminal repeat (MMTV LTR) having a glucocorticoid response element (GRE), enhancer sequences from simian virus 40 (SV40) and herpes simplex virus type 1 (HSV-1), human cytomegalovirus (hCMV), thymidine kinase (Tk), and locus control regions of the metallothionein gene (LTR-MT) displaying variable potencies in driving transcription (Blackwood and Kadonaga 1998; Makrides 1999).
Both transient and stable expression of genes in mammalian cells have many advantages. First, genes are expressed in a native environment so post-translational modifications and subcellular targeting are authentic. Second, many transformed mammalian cell types are available for transfection. This allows for the selection of cell types—with the proper intracellular accessory factors or proteins—for enzyme activities that most closely resemble their in vivo counterparts. Finally, expression of gene products can be controlled by an increasing number of eukaryotic promoters. Thus, the levels of expression, including inducible expression, can be controlled.
Generations of cell lines that stably express DME genes represent the new generation of pharmacological and toxicological test systems (Langenbach et al. 1992). Expression of stably transfected DME genes in Epstein-Barr virus (EBV)-transformed human B-lymphoblastoid cell lines has provided cell lines that scientists can use to study and categorize numerous foreign compounds. Those drugs or chemicals can be classified according to the intermediates formed by way of the different metabolic pathways. More than a dozen EBV-transformed human B-lymphoblastoid cell lines are already commercially available (Crespi and Miller 1999). They contain anywhere from 1 to 12 human P450 and other DME genes (i.e., cDNAs), which retain their substrate specificity with regard to the metabolism of particular drugs or classes of pharmaceutical agents.
Some of these cell lines are already being used by pharmaceutical companies to determine whether a newly developed drug is metabolized by a particular DME. If, for example, the new drug is shown to be a CYP2D6 substrate, it is already known that humans differ by 10-fold to more than 30-fold in the activity of that enzyme (see Chapter 5). Poor metabolizers (the PM phenotype, which comprises 6% to 10% of Caucasian populations) would thus be expected to metabolize the new drug more slowly than extensive metabolizers (EM phenotype) and much more slowly than ultra-metabolizers (UM phenotype). If the parent drug is toxic, the PM individual would be at increased risk; if the CYP2D6-mediated metabolite is toxic, then the EM and especially the UM individual would be at increased risk. Dosage of the drug can therefore be adjusted to the patient’s genotype before the physician prescribes the new drug. For drugs already on the market, molecular epidemiologists can search for possible associations between reported differences in birth defects or other developmental problems and genotype of the