High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds

We have used transgenic tobacco seeds to produce large amounts of a functionally active engineered antibody. A gene infusion encoding an antigen-binding single chain Fv protein (scFv) that recognizes the hapten oxazolone was constructed and used as a model. After characterization in a bacterial expression system ,the scFv gene was cloned into a plant expression cassette conferring seed specific expression, and transferred using Agrobacterium-mediated transformation, into Nicotiana tabacum. The expressed scFv could be detected in the developing as well as ripe seeds of regenerated transgenic plants, and the functionally active scFv is stabaly deposited and accumulates up to 0.67% of the total soluble seed protein. After storage of ripe transgenic tobacco seeds for one year at room temperature there was no loss of scFv protein or its antigen-binding activity.     

Analysis of the human ferrochelatase promoter in transgenic mice

Ferrochelatase catalyzes the chelation of ferrous iron and protoporphyrin to form heme. It is expressed as a housekeeping gene in all cells, but is upregulated during erythropoiesis. Ferrochelatase activity is deficient in the inherited disease protoporphyria as a result of heterogeneous mutations. Although human ferrochelatase is transcribed from a single promoter in both nonerythroid and erythroid cells, previous studies using transient transfection assays failed to demonstrate erythroid-specific increased expression from 4.0 kb of the human ferrochelatase promoter containing the erythroid cis-elements, GATA and NF-E2. The present study analyzes the in vivo regulation of the ferrochelatase gene to provide insights into the mechanism of its erythroid-specific enhancement. Transgenic (TG) mouse lines were generated in which the luciferase reporter gene was driven by either a 150-bp ferrochelatase minimal promoter (-0.15 TG) or by a 4.0 kb extended 5' upstream region (-4.0 TG). Expression of the -4.0 TG transgene was generally consistent with the endogenous gene during embryonic development and in nonerythroid and erythroid tissues as demonstrated by Northern blotting and mRNA in situ hybridization. The -4.0 TG was expressed at a higher level than the -0.15 TG in nonerythroid and erythroid tissues, including during extramedullary erythropoiesis induced by n-acetylphenylhydrazine injection. The enhanced erythroid expression of the -4.0 TG correlates with the appearance of a DNase I hypersensitive site in the 5' flanking region of the transgene. Therefore, in the context of chromosomal integration, the 5' flanking region of the ferrochelatase gene is necessary and sufficient to confer high levels of transgene expression in erythroid tissue.     

Expression of rat bone sialoprotein promoter in transgenic mice

Bone sialoprotein (BSP) is a major protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite crystals. Our previous studies have demonstrated that BSP mRNA is expressed by differentiated osteoblasts, odontoblasts, and cementoblasts involved in de novo mineralized tissue formation in a tissue-specific and developmentally regulated manner. To determine the basis of the selective expression of the BSP gene, we have generated four transgenic mouse lines in which 2.7 kb of the rat BSP promoter ligated to a luciferase reporter gene has been stably integrated into the mouse genome. Assays of luciferase activities in 5-day-old animals has revealed consistently high levels in bone tissues with negligible activities in various other organs including kidney, liver, stomach, intestine, and spleen. In some animals, variable expression was observed in brain and skin. Temporal analyses revealed the highest luciferase expression in neonatal bones, with expression decreasing markedly with subsequent growth and development, as observed previously for the endogenous gene in rats. Immunohistochemical analysis of luciferase activity and in situ hybridization of luciferase mRNA in bone tissues show that differentiated osteoblasts express the highest levels of luciferase, consistent with the induction of endogenous gene expression. These studies demonstrate that the regulation of the BSP gene during osteoblastic differentiation, together with its tissue-specific, developmentally regulated expression, is primarily mediated within the 2.7 kb region of the promoter.     

Expression of de novo high-lysine alpha-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants

Transgenic insect technology will provide opportunities to explore the basic biology of a broad range of insect species in ways that will prove insightful and important. It is also a technology that will provide opportunities to manipulate the genotypes of insects of practical significance to the health and welfare of humans. The Hermes transposable element from the housefly, Musca domestica, is a short inverted repeat-type element related to hobo from Drosophila melanogaster, Ac from Zea mays, and Tam3 from Antirrhinum majus. It has potential to become a versatile and efficient broad host-range insect transformation vector. The ability of Hermes to transpose when introduced into five species of diptera from four divergent families was tested using an in vivo, interplasmid transpositional recombination assay. Hermes was capable of transposing in all species tested, demonstrating that Hermes has a broad host-range. In addition, the rates of transposition were sufficiently high in all species tested to suggest that Hermes will be an efficient gene transfer vector in a wide range of insect species. The Hermes element also revealed a pattern of integration into the target substrate that permitted factors determining integration site selection to be identified. Primary nucleotide sequence of the integration site played a role as did proximity to preferred integration sites and the nucleosomal organization of the target.     

Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.     

Colocalization of barley lectin and sporamin in vacuoles of transgenic tobacco plants

So-called runting stunting syndrome (RSS) afflicts chicks worldwide. The present study in Georgia chicks is the first report of unique histologic features of RSS pathology in chicks in the United States. Various combinations of avian nephritis virus, enterovirus-612, and reovirus were always isolated from chick small intestines. Ultrastructurally, only small round viruses were seen in small intestinal lesions. Although finding intralesional virus in small intestinal segments from chicks with signs and gross lesions consistent with RSS constitutes a reasonable criterion for making a diagnosis of this disease, chicks without intralesional viruses and with bacterial or protozoal enteritis may also be small and abnormally feathered. Because just what constitutes RSS remains a diagnostic dilemma, we recommend that the use of the imprecise acronym "RSS" be discontinued.     

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the beta-glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.     

Temporal and spatial specificity of PDGF alpha receptor promoter in transgenic mice

Aberrant expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) has been linked to developmental abnormalities in vertebrate models, and has been implicated in multiple disease states in humans. To identify cis-acting regulatory elements that dictate expression of this receptor, we generated transgenic mice bearing the reporter gene beta-galactosidase (lacZ) under the control of a 6-kb promoter sequence. Expression of lacZ was monitored throughout embryonic development, with special focus on nervous tissue, skeleton, and several organ systems wherein PDGF alpha R expression is thought to play a pivotal role. In several independent transgenic mouse strains, lacZ expression recapitulated predominant features of PDGF alpha R gene expression during mouse development. These results demonstrate that critical tissue-specific regulatory elements for PDGF alpha R expression are located within a 6-kb upstream region of the PDGF alpha R gene.     

Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis

To determine the regulatory mechanism of gene expression in the early stages of tracheary element (TE) differentiation, we isolated and characterized a genomic fragment of TED3 whose mRNA is expressed preferentially in differentiating TEs 12-24 h before morphological changes in the in vitro Zinnia system. Transgenic Arabidopsis plants with a chimeric gene of the 537 bp TED3 promoter and the beta-glucuronidase (GUS) reporter gene indicated the strong expression of the GUS gene by the TED3 promoter in TEs, in particular in immature TEs as well as stipules and trichomes. GUS expression driven by the promoter was also induced in callus, in which GUS activity was localized in immature TEs and slender cells around TEs that may be TE precursor cells. The TED3 promoter was not significantly activated by wounding. This pattern of expression differed clearly from that of other vascular tissue-related genes such as PAL, 4CL, and GRP1.8. The nature of TED3 promoter enables us to use it to monitor TE differentiation in tissue and to introduce foreign genes preferentially into immature TE.     

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.     

Sequencing and functional analysis of the SNRPN promoter: in vitro methylation abolishes promoter activity

The gene encoding the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) maps to the Prader-Willi syndrome critical region on chromosome 15 and is expressed preferentially from the paternal allele. A CpG island encompassing the first exon of SNRPN is methylated on the inactive maternal allele. DNA sequence was determined for a cosmid containing the first three exons of SNRPN and extending 20 kb upstream and 15 kb downstream from the CpG island. This region is extremely rich in Alu elements and other repetitive sequences and contains a single CpG island, which includes numerous short direct repeat sequences. Functional analysis of the first exon revealed strong promoter activity for a 260-bp fragment extending 207 bp upstream from the exon. In vitro methylation of this 260-bp fragment abolished promoter activity completely, suggesting that the silencing of the maternal SNRPN allele may be a direct consequence of methylation of the promoter region.