The ROSA␤geo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and f luorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral ␤geo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5 ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans.

We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli fi-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescenceactivated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for 13-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of f-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescenceactivated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of (3-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.The Escherichia coli lacZ gene has previously been adapted for use in mammalian cells and has shown utility as a marker for the expression of chimeric genes (1, 2). In these previous studies, the chromogenic f3-galactoside analog o-nitrophenyl /3-D-galactopyranoside (ONPG) was used as an alternative to the chloramphenicol acetyltransferase assay (3) to quantitate gene expression from heterologous promoters fused 5' proximal to the lacZ structural gene. However, in both the chromogenic LacZ and radioactive chloramphenicol acetyltransferase assays, extracts of bulk populations of cells are used to quantitate an average promoter strength/enzyme activity and, thus, do not give any indication of the heterogeneous expression patterns that could exist within complex cell populations. These other assays also do not provide one with a means to select viable cells expressing known quantities of gene product.lacZ has also proven effective as a marker in the genetic tagging of cell lineage founders and subsequent tracing of progeny in tissue sections using a cytochemical stain for expression of LacZ (4, 5). Using recombinant, replicationdefective Moloney murine leukemia viruses (Mo-MuLV) in which the expression of lacZ is permitted from an internal simian virus 40 (SV40) promoter, Sanes et al. (4) Here, we use the f3-galactoside analog fluorescein di-,B-Dgalactopyranoside (FDG) (11) in a protocol, termed " 'ACS-FDG," that sensitively distinguishes LacZ' cells from LacZ-cells and allows time-dependent fluorescenceactivated cell analysis and sorting. FDG is cleaved by 03-galactosidase in LacZ' cells to yield fluorescein, which can be detected by FACS. We find that the kinetics of fluorescence accumulation within LacZ' cells, after FDG substrate loading, shows a linear dependence over time until maxima are approached because of...

Cyclosporin A and FK506 are immunosuppressive compounds that have similar inhibitory effects on the expression of several lymphokines produced by T lymphocytes. Despite their similar effects the drugs bind to two different cytosolic protein, cyclophilin and FKBP respectively, which raises the possibility that they have different modes of action. Using constructs in which mRNA production controlled by a specific transcription factor could be readily measured we found that both cyclosporin A and FK506 completely inhibited transcription activated by NF‐AT, NFIL2 A, NFIL2 B and partially inhibited transcription activated by NF kappa B. Cyclosporin A and FK506 inhibited only transcriptional activation that was dependent on Ca2+ mobilization. However, cyclosporin A and FK506 did not inhibit Ca2+ mobilization dependent expression of c‐fos mRNA indicating that only a subset of signalling pathways regulated by Ca2+ is sensitive to these drugs. Furthermore, we did not observe any qualitative differences between the effect of cyclosporin A and FK506 on six different transcription factors which suggests that these drugs may interfere with the activity of a novel Ca2+ dependent step that regulates several transcription factors.

We have studied enhancer function in transient and stable expression assays in mammalian cells by using systems that distinguish expressing from The mechanism by which enhancers activate transcription remains a subject for speculation. One proposed mode of enhancer action is to increase the rate of transcription from a linked promoter (Fig. 1A). This "rate" model is based on nuclear run-on assays of populations of cells transiently transfected with plasmid constructs, which found that more nascent transcripts were synthesized by cells if the transfected construct contained the simian virus 40 (SV40) enhancer (1, 2).The interpretation of this result as an increase in the rate of transcription in every transfected nucleus, or an increase in the density of RNA polymerase on each template (3, 4), underlies the assessment of much subsequent work on transcriptional control. A "probability" model of enhancer action (Fig. 1B) has been suggested by experiments that have examined single cells, rather than populations, expressing transfected constructs (5-7). These experiments revealed that enhancers increase the number of expressing cells but not the level of expression per expressing cell.The results of the nuclear run-on experiments cited above are actually consistent with both models, since either an increase in the number of expressing cells in the population or an increase in the polymerase density per individual template would yield an increase in nascent transcripts in the total cell population. However, only the probability model is consistent with the single cell experiments.We have made a detailed examination of the enhancer effect on expression of a linked reporter gene using both transient and stable expression assays. These experiments were designed to directly test the two models by distinguishing and separating expressing from nonexpressing transfected cells. We find that in both transient and stable assays a linked enhancer increases the number of cells actively expressing a reporter but not the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.A "Rate" B "Probability"