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Bacterial ,B-galactosidase, encoded by the lacZ gene, serves as a sensitive cytochemical marker in eukaryotic cells and tissues. In transient expression experiments, human and simian cells stain blue 48 hr after transfection with a plasmid containing a lacZ gene, whose expression is directed by a simian virus 40 promoter containing a synthetic lactose operator sequence. Transfection efficiency was about 0.6%. Incorporation of an operator sequence within the promoter permits regulation of .3-galactosidase gene expression by the lad gene product, the lac repressor. When cells were cotransfected with the lacZ plasmid and a second plasmid containing the lacI gene, /8-galactosidase activity was extinguished. Its activity could be reestablished to original levels upon application of isopropyl 8-D-thiogalactoside to transfected cells. A cell line that stably carries both the lacI and lacZ genes was efficiently induced to synthesize fi-galactosidase after isopropyl i3-D-thiogalactoside administration. In transient expression experiments and in stably transfected lines, repression and induction of 8-galactosidase activity were predominantly at the transcriptional level.The ability to dictate the reversible expression and repression of specific eukaryotic genes represents a potent tool for studying cellular activity. Efforts to this end have primarily utilized the mouse mammary tumor virus promoter or the mouse metallothionein promoter fused to a heterologous gene or cDNA (1-7). In the former case, administration of glucocorticoid, which binds a glucocorticoid receptor, results in gene activation. The ligand-receptor complex interacts with an enhancer-like glucocorticoid receptor element in the mouse mammary tumor virus promoter, thereby stimulating transcriptional activity. In the latter case, gene activation is induced by exposure to heavy metals. Although both systems have been used with variable success, they are limited by restrictions in tissue-type expression and in substantial background levels of expression in cell types that permit induction. Other inducible promoter systems that have been used to achieve regulated gene expression, but with similar reservations, include heat-shock-inducible promoters (8, 9) and poly(IC)-inducible promoters (10).The concept of using bacterial regulatory systems in mammalian cells has numerous attractive features. These systems have the potential for imposing very stringent regulation in a tissue-independent manner. There is also a reduced likelihood that prokaryotic regulatory proteins will interact with host DNA sequences that are similar to target sequences, in a biologically meaningful fashion. One concern in adapting a prokaryotic regulatory system to a mammalian nuclear milieu is whether or not prokaryotic regulatory proteins can access DNA in chromatin efficiently and with specificity. This concern is relieved by the demonstration that the cre-lox site-specific recombination system of coliphage P1 can operate efficiently in yeast (11). The cre recombinase is able to...

Delivery and expression of the herpes simplex virus thymidine kinase (HSVtk) gene in combination with the prodrug ganciclovir is currently being evaluated for the treatment of many types of cancer. After initial phosphorylation by HSVtk, cellular kinases generate the toxic triphosphate form of ganciclovir (GCV). To further define the role of GCV metabolism in cells expressing HSVtk, two human tumor cell lines, UMSCC29 and T98G, were transduced with HSVtk and screened for insertion of one or two copies of the viral transgene by Southern blot analysis. Both the relative capacities for incorporating labeled GCV and the levels of GCV metabolites were determined for each of the parental cell lines and their derivatives containing either one or two copies of the HSVtk gene. The efficiency of GCV killing and the magnitude of the bystander effect were compared for the single-and double-copy HSVtk cell lines. Consistently, cells that expressed two copies of HSVtk metabolized GCV more efficiently, were more sensitive to GCV, and demonstrated improved bystander killing relative to single-copy HSVtk cells. The implications of these results for future and current therapies employing HSVtk and GCV are discussed. Cancer Gene Therapy (2000) 7, 240 -246

In theory, sunshine exposure is sufficient to maintain normal vitamin D concentrations for the optimal growth of newborn infants. To determine whether season of birth, latitude (north v. south) and increasing dosages of vitamin D supplements would influence the growth rate for the first 6 months of life, 255 healthy fall-and spring-born infants from two northern and two southern cities in China were randomly assigned to receive either 100, 200, or 400 IU of vitamin D a day. The study showed that season of birth and dose of vitamin D did not affect the growth rate of infants born in the same latitude, but a significant difference was found in the gain in length over the 6-month period between infants from the north and infants from the south (P = 0.0001). Regional differences among the Chinese people, other than sunshine exposure, may have influenced the difference in length gain.