The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.     

Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos

To generate stable lines of transgenic fish, early zebrafish embryos were injected with high concentrations of a linear bacterial plasmid. After injection, the foreign DNA was converted into a high molecular weight form and then amplified approximately tenfold during the initial rapid cleavages characteristic of the early embryo prior to gastrulation. While most of this DNA was subsequently degraded during gastrulation, some of the foreign sequences survived the gastrula stage and could be found in most of the injected fish at 3 weeks of age. Only about 5% of fish analysed 4 months after the injection retained foreign DNA in their fins, usually at less than one copy per cell. One of these fish was also found to contain about 100 copies per cell of foreign DNA in a fraction of its germ cells. Approximately 20% of the F1 offspring from this germ-line-positive parent inherited the foreign DNA, whereas 50% of F2 progeny obtained from an identified F1 individual inherited these sequences. The 50% transmission rate in F2 progeny was as expected for a single, heterozygous genomic insert. These observations indicate that injected DNA can be integrated into the fish genome, that the resulting transgenic fish are mosaic and that some of these mosaic individuals give rise to stable lines of transgenic fish.     

Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression

To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.     

Comparison of the activity of carp and rat β-actin gene regulatory sequences in tilapia and rainbow trout embryos

A comparative study on the level of expression of lacZ reporter constructs driven by equivalent carp and rat β-actin regulatory sequences was carried out in embryos of tilapia and rainbow trout. DNA was microinjected into fertilised tilapia and rainbow trout eggs and the embryos/fry were assayed at various developmental stages for β-galactosidase expression. We provide evidence to demonstrate that the carp β-actin promoter/lacZ reporter gene is expressed at higher levels than the equivalent rat β-actin construct in both species. © 1996 Wiley-Liss, Inc.     

Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualizing dynamic E2F-mediated repression in vivo

Although many E2F target genes have been identified recently, very little is known about how any single E2F site controls the expression of an E2F target gene in vivo. To test the requirement for a single E2F site in vivo and to learn how E2F-mediated repression is regulated during development and tumorigenesis, we have constructed a novel series of wild-type and mutant Rb promoter-LacZ transgenic reporter lines that allow us to visualize the activity of a crucial E2F target in vivo, the retinoblastoma tumor suppressor gene (Rb). Two mutant Rb promoter-LacZ constructs were used to evaluate the importance of a single E2F site or a nearby activator (Sp1/Ets) site that is found mutated in low-penetrance retinoblastomas. The activity of the wild-type Rb promoter is dynamic, varying spatially and temporally within the developing nervous system. While loss of the activator site silences the Rb promoter, loss of the E2F site stimulates its activity in the neocortex, retina, and trigeminal ganglion. Surprisingly, E2F-mediated repression of Rb does not act globally or in a static manner but, instead, is a highly dynamic process in vivo. Using neocortical extracts, we detected GA-binding protein alpha (GABPalpha, an Ets family member) bound to the activator site and both E2F1 and E2F4 bound to the repressor site of the Rb promoter in vitro. Additionally, we detected binding of both E2F1 and E2F4 to the Rb promoter in vivo using chromatin immunoprecipitation analysis on embryonic day 13.5 brain. Unexpectedly, we detect no evidence for Rb promoter autoregulation in neuroendocrine tumors from Rb+/-; RbP-LacZ mice that undergo loss of heterozygosity at the Rb locus, in contrast to the situation in human retinoblastomas where high RB mRNA levels are found. In summary, this study provides the first demonstration that loss of an E2F site is critical for target gene repression in vivo and underscores the complexity of the Rb and E2F family network in vivo.     

Heat-inducible expression of a reporter gene detected by transient assay in zebrafish

Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system. Cells expressing beta-galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene. Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35-36 degrees C up to 39 degrees C with an apparent decrease at 40 degrees C), resembling that of the zebrafish hsp70 genes. However, their induction kinetics were different, which might be due to differences in their 5' UTRs. Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level. These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish.     

Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins. It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation. We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC). Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the Escherichia coli beta-galactosidase coding sequence were generated. Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins. The anatomic locations of beta-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus. Trigeminal ganglion neurons were positive for beta-galactosidase in adult ICP0 and ICP27 reporter transgenic mice. The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age. beta-galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody. Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for beta-galactosidase labeling in the absence of HSV-1. We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins. These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons.     

Multiple CArG boxes in the human cardiac actin gene promoter required for expression in embryonic cardiac muscle cells developing in vitro from embryonal carcinoma cells.

Chimeric genes composed of the human cardiac actin promoter driving the Escherichia coli lacZ reporter gene were constructed, transfected, and stably integrated into genomes of P19 embryonal carcinoma cells. The transfected constructs were expressed actively in cardiac myocytes formed following dimethyl sulfoxide (DMSO)-induced cell differentiation but poorly in undifferentiated cultures and in cultures treated with retinoic acid to develop into derivatives of the neuroectoderm. A number of deletions of the promoter were constructed and tested. Three regions required for efficient expression in P19-derived cardiac muscle were identified, each containing sequences referred to as CArG boxes (CC[AT-rich]6GG). This analysis indicated that regulatory sequences important for expression in cardiac muscle were present upstream of the core promoter identified previously by transient assays in skeletal myoblasts. Expression of the cardiac actin promoter was enhanced 10-fold in undifferentiated P19 cells in the presence of the myoD protein. The promoter regions important for expression in P19-derived cardiocytes were similar to those important for myoD-induced enhancement, a result we interpret to be consistent with the idea that cardiac muscle might contain a myoD-like activity.     

Intron 1 sequences are required for pancreatic expression of the human proglucagon gene

The mammalian proglucagon gene is expressed in pancreatic islet A-cells, intestinal L-cells, and select neurons of the brain, where posttranslational processing results in the liberation of a unique profile of peptides. Despite the importance of proglucagon-derived peptides in human biology, little is known about the regulation of the human gene, as the rat gene has been the preferred model for understanding the regulation of proglucagon gene expression. Previously, we have shown that although the immediate promoter region of the rat proglucagon gene is sufficient for expression in pancreatic islet cells, the homologous human proglucagon promoter sequences are not sufficient. We have now used a comparative genomic approach to identify noncoding sequences near the human proglucagon gene that are conserved among mammals, and thus potentially are regulatory sequences. Our alignments identified three evolutionarily conserved noncoding regions (ECR), one is the immediate promoter region (ECR1), the second is about 5 kb 5' to the mRNA start site (ECR2), and the third is near the 3' end of the first intron (ECR3). Our in vitro transient transfection assays with reporter gene constructs that include the human ECR3 support expression in rodent islet cell lines. Complementary studies with transgenic mice possessing a reporter gene regulated by a human proglucagon gene promoter-intron 1 (including ECR3) sequences express the reporter gene in the pancreas, as well as the intestine and selected neurons. These studies suggest that conserved sequences within intron 1 of the human proglucagon gene are important for expression in the pancreas.     

Generating lineage-specific markers to study Drosophila development.

To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of beta-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.