Codon optimization markedly improves doxycycline regulated gene expression in the mouse heart

Tetracycline regulated gene expression in transgenic animals is potentially a very powerful technique (Furth et al., 1994; Gossen & Bujard 1992). We have utilized this system in an attempt to overcome the perinatal lethality resulting from constitutive transgenic expression in the heart (Valencik & McDonald, Am J Physiol Heart Circ Physiol 280: H361–H367). We found that compound hemizygous animals created by mating selected reverse tetracycline transactivator (rtTA) and transresponder (TR) lines display tightly regulated TR expression in the heart. However, we identified two fundamental problems. First, codon usage bias appeared to severely limit the expression of the rtTA driven by the cardiac -myosin heavy chain promoter. Second, co-injection of rtTA and TR transgenes led to compound hemizygous animals that exhibited unregulated TR gene expression. Codon optimization of the rtTA construct leads to marked improvement (increasing the average induction from 20-fold to 832-fold) in cardiac myocyte expression. The resulting opt-rtTA lines can be bred to homozygosity, facilitating rapid screening of F0 TR animals for doxycycline regulated transgene expression.     

Reporter genes in transgenic mice

Althoughin vivo models utilizing endogenous reporter genes have been exploited for many years, the use of reporter transgenes to dissect biological issues in transgenic animals has been a relatively recent development. These transgenes are often, but not always, of prokaryotic origin and encode products not normally associated with eukaryotic cells and tissues. Some encode enzymes whose activities are detected in cell and tissue homogenates, whereas others encode products that can be detectedin situ at the single cell level. Reporter genes have been used to identify regulatory elements that are important for tissue-specific gene expression or for development; they have been used to producein vivo models of cancer; they have been employed for the study ofin vivo mutagenesis; and they have been used as a tool in lineage analysis and for marking cells in transplanation experiments. The most commonly usedin situ reporter gene islacZ, which encodes a bacterial -galactosidase, a sensitive histochemical marker. Although it has been used with striking success in cultured cells and in transgenic mouse embryos, its postnatalin vivo expression has been unreliable and disappointing. Nevertheless, the ability to express reporter genes in transgenic mice has been an invaluable resource, providing insights intoin vivo biological mechanisms. The development of newin vivo models, such as those in which expression of transgenes can be activated or repressed, should produce transgenic animal systems that extend our capacity to address heretofore unresolved biological questions.     

Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice

The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human beta-actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16-responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre-implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2-cell to the 4-cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility.     

Cis effect oflacZ sequences in transgenic mice

Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving theEscherichia coli -galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression inHMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of doubleHMG-lacZ andHMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that thelacZ sequence might interfere negatively with the expression of the adjacentHMG-cat transgene.     

Conditional Transgene Expression in Endothelial Cells

The ability to tightly control transgene expression in vivo provides an opportunity to determine the role of certain gene products at different times during development and/or in response to different stimuli. We have characterized and evaluated a tetracycline-responsive endothelial-specific binary system during mouse development, by engineering several transgenic lines which drive the expression of a tetracycline- controlled transactivator (tTA) under the control of either the Tek or Tie promoters (driver lines). We have also generated a responder line which carries multiple copies of the tTA DNA binding element (tetos) upstream of a reporter gene coding for a nuclear targeted -galactosidase (responder lines). No expression of the target transgene was detected in mice homozygous for the reporter transgene. On mating the driver lines with the responder line, expression of -galactosidase from the reporter transgene was detected within the endothelium. Responder transgene expression was repressed rapidly upon addition of doxycycline to the drinking water. Importantly, this repression was reversible upon withdrawal of the drug. This approach should be useful to deliver the expression of potentially toxic gene products or rescue embryonic mutations that affect either the endothelial lineage or production of growth factors which are secreted systemically.     

Visualization and ablation of phenylethanolamineN-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5 flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli -galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT_{8 kb}-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.     

Agrobacterium-mediated transformation of white mustard (Sinapis alba L.) and regeneration of transgenic plants

Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and -glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.Abbreviations BAP 6-benzylaminopurine - CaMV cauliflower mosaic virus - GUS -glucuronidase - IBA indole-3-butyric acid - IM infection medium - NAA 1-naphthalene acetic acid - neo gene encoding NPTII - NPTII neomycin phosphotransferase - RIM root-inducing medium - SEM shoot-elongation medium - SIM shoot-inducing medium - t-nos polyadenylation site of the nopaline synthase gene - uidA gene encoding GUS - WM wash medium - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide     

Expression and inheritance of foreign genes in transgenic peanut plants generated byAgrobacterium-mediated transformation

To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T₂ plants gave either 100% or 3:1 expression of the -glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T₁ plants. Fluorometric GUS assay in T₁ and T₂ generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.Abbreviations GUS -Glucuronidase - MS Murashige and Skoog - MU 4-Methylumbelliferone - NPTII Neomycin phosphotransferase II     

Tet-system for the regulation of gene expression during embryonic development

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTA ^CMV (M1) and rTA ^CMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and -galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTA ^CMV (M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTA ^CMV -3/NZL-2 embryos at E13.5. Doxycycline abolished -gal expression in tTA ^CMV (M1)/NZL-2 but induced it in rTA ^CMV -3/NZL-2 embryos including late stages of embryogenesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that double reporter animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.     

Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression

Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo ^r ) and thelacZ gene which codes for intracellular -galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and -galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneo ^r gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneo ^r gene expression, must be negligible, as higher expression of -galactosidase (1.5×10^–6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10^–7 units/cell). Thus, the main factor causing severe growth reduction (metabolic burden) in cells containing the amplifieddhfr gene system was not overexpression of -galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.     

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of -galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. -galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial–mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.     

Cytoplasmic actin A3 gene promoter injected as supercoiled plasmid is transiently active inBombyx mori embryonic vitellophages

Summary Freshly deposited eggs ofBombyx mori were microinjected with supercoiled plasmid DNA which carried the -galactosidase coding sequence ofEscherichia coli inserted in place of the coding sequence of theB. mori cytoplasmic actin A3 gene. Transient expression of this fusion gene in the embryo was determined by in situ histochemical detection of enzyme activity. After injection of the plasmid at different stages of embryonic development, -galactosidase activity depending on the injected DNA was only detected in the vitellophages. This indicates the presence of active transactivators of the actin A3 gene promoter in this cell type. Tissue specificity of the fusion gene expression could be related to the early polyploidization of vitellophages, a process which would favour the stability of the nuclear pool of injected plasmids. The activity of the transgene in vitellophages was detectable at 24–33 h of egg development, the stage presumed for the onset of zygotic gene expression, up to the end of embryogenesis. This gene transfer system is thus promising to analyse thecis regulatory sequences of the actin A3 gene and could be utilized for other ubiquitous genes. Correspondence to: M. Coulon-Bublex     

The Use of the Uromodulin Promoter to Target Production of Recombinant Proteins into Urine of Transgenic Animals

A uromodulin promoter has been isolated, sequenced, and used to generate two sets of transgenic mice for expression of the lacZ marker gene and for production of the human recombinant erythropoietin (rhEPO) in urine. We demonstrated that the 5.6-kb fragment of the uromodulin gene containing the 3.7-kb promoter area and, both the first exon and part of the second exon, were sufficient to provide kidney-specific expression of the lacZ gene. Histological analysis of the lacZ expression pattern revealed -galactosidase activity specifically in the thick limb of Henle's loop. However, due to random integration of the transgene, ectopic expression was detected in some transgenic lines. Analysis of the EPO-transgenic mice showed that rhEPO was secreted into the urine of founder mice (up to 6 ng/ml). We were able to breed and analyze only two sublines with a very low expression level of rhEPO (up to 260 pg/ml). All of our transgenic mice expressing rhEPO in urine developed disease symptoms similar to polycythemia in humans. These included a considerable increase in red blood cell counts, hemoglobin concentration, and hematocrit concomitant with severe thrombocytopenia, all of which were detected in the rhEPO-expressing mice. Although our model did not prove to be beneficial for commercial production of rhEPO, we concluded that the uromodulin promoter could be useful for expression of other important therapeutic proteins into the urine of transgenic animals.     

A quick and efficient system for antibiotic-free expression of heterologous genes in tobacco roots

Requirement for antibiotic-resistance selection markers and difficulty in identifying transgenes with the highest expression levels remain the major obstacles for rapid production of recombinant proteins in plants. An alternative approach to producing transgenic plants free of antibiotic-resistance markers is the phenotypic-based selection with root-proliferation genes (rol genes) of Agrobacterium rhizogenes. By using Agrobacterium tumefaciens harboring the pRYG transformation vector with a cluster of rol genes linked to a heterologous gene of interest, we have developed a rapid transformation tool using hairy root formation as a selection marker. The expression of -glucuronidase in newly induced transgenic tobacco roots could be detected as early as 12 days after inoculation. Higher levels of transgene expression in the roots correlated positively with the rates of root elongation on hormone-free medium and thus could be used for positive selection. When tobacco plants were transformed with pRYG harboring the expression cassette for secreted alkaline phosphatase (SEAP), the release of SEAP from roots of the fully regenerated transgenic plants could be quantified at rates as high as 28 g/g root dry weight per day.Abbreviations GUS: -Glucuronidase - SEAP: Secreted alkaline phosphatase - rolABC: Cluster of rolA, rolB, and rolC genesCommunicated by A. Altman     

Down regulation of the mercury resistance operon by the most promoter-distal gene merD

Summary The effect of the merD gene on the expression of the mer operon was determined from the rates of accumulation of merA-lacZ fusion protein in the presence and absence of an active merD gene in trans. In the presence of the merD gene, -galactosidase activity was 2- to 4-fold lower. The merD gene was cloned in a T7 promoter expression vector and the MerD protein product was visualized by autoradiography.     

Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia

Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.     

β-Galactosidase deficiency in juvenile and adult patients

Summary Six juvenile and adult patients with progressive neurological diseases and -galactosidase deficiency were reported. Any diseases known to date were denied. These cases together with ten case reports in the literature were reviewed and were classified into three groups from clinical and biochemical points. Group 1 patients were characterized by progressive ataxia and myoclonus with gargoyle changes and macular cherry-red spots. In this syndrome -galactosidase activity seems to be secondarily affected by other biochemical defects. A group 2 patient showed similar neurological manifestations without gargoyle changes or macular cherry-red spots. Patients with these clinical features not associated with -galactosidase deficiency have also been described in the literature. Group 3 patients had progressive pyramidal and extrapyramidal disease without gargoyle changes or macular cherry-red spots. These cases may represent juvenile and adult type G_M1-gangliosidosis. Accumulation of G_M1 has not yet been demonstrated.