ABSTRACT
Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Escherichia coli Proteins , Genetic Vectors/genetics , Mice, Transgenic/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Animals , Bacterial Proteins/drug effects , Base Sequence , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cells, Cultured , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Cloning, Molecular/methods , Dose-Response Relationship, Drug , Eukaryotic Cells , Female , Fetus/cytology , Fetus/metabolism , Gene Expression Regulation , Genes, Reporter , Genetic Vectors/metabolism , Isopropyl Thiogalactoside/pharmacology , Lac Repressors , Male , Methylation , Methylgalactosides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/drug effects , Thiogalactosides/pharmacology , Time Factors , Tissue DistributionABSTRACT
Macrophages and other cells are capable of ingesting a variety of solids from their external environment. When such phagocytic processes occur in animals, they can lead to phagocytosis from the respiratory or the digestive tract of particles containing minute air emobli that may serve as bubble nuclei upon exposure of the animal to conditions of gas supersaturation. To test whether this is possible, gas supersaturation tolerances were determined for murine macrophages and macrophage-like tumor cells, and for cells of the slime mold Dictyostelium discoideum, before and after phagocytosis of particles that were effective in inducing bubble formation in nitrogen-supersaturated aqueous suspensions. After phagocytosis, the ability of the particles to induce bubble formation was completely abolished. All three cell types essentially retained their normal high resistance to bubble formation; even nitrogen supersaturations in excess of 150 atm (1.55 x 10(7) Pa) did not lead to internal bubbles. Alterations of the particle surfaces and unique properties of the intracellular fluid appear to be the underlying cause of the extremely high gas supersaturation tolerances observed.
