Levels Of Transient Expression Research Articles

Regulation of Synaptophysin Degradation by Mammalian Homologues of Seven in Absentia

Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin.

Effects of cetyltriethylammonium bromide on the replication of Bombyx mori nucleopolyhedrovirus.

An experimental study was undertaken to quantify the effects of cetyltriethylammonium bromide (CTAB) on the replication of Bombyx mori nucleopolyhedrovirus (BmNPV) and the transcriptionalactivity of BmNPV ie-1 promoter. The results demonstrated that the budded virus (BV) titer rose about 3.7-fold by adding CTAB to the culture media up to 0.1 mu g ml(-1) in infected Bm-N cells with a wild-type BmNPV. The transient expression level of luciferase driven by BmNPV ie-1 promoter was enhanced by more than 3-fold in the presence of 0.1 mu g ml(-1) of CTAB in uninfected insect cells via a transient expression system. Contrary to the rise in BV titer, the polyhedra inside the nucleus of infected cells dropped linearly from 4.0 x 10(6) ml(-1) down to 2.1 x 10(6) ml(-1) with in a range of CTAB concentrations from 0 to 0.25 mu g ml(-1). The same trend in expression level of beta -galactosidase or phytase was given when the Bm-N cells or fifth-instar silkworm larvae infected with a recombinant BmNPV containing the beta -galactosidase or phytase reporter gene driven by the polyhedrin promoter. We deduced that CTAB appeared to affect the virus bi-phasic life cycle stages and production pathways, resulting in an enhancement in BV production and a suppression of occluded virus (OV) production and expression of foreign genes controlled by the polyhedrin promoter.

Cross‐linked low molecular weight glycopeptide‐mediated gene delivery: Relationship between DNA metabolic stability and the level of transient gene expression in vivo

DNA co‐condensates were formed by reacting [125I]DNA with an admixture of a high‐mannose glycopeptide (Man9‐CWK18) and either of two poly(ethylene glycol) peptides (PEG‐VS‐CWK18 or PEG‐SS‐CWK18) followed by cross‐linking with 6–50 mol equiv of glutaraldehyde. [125I]DNA co‐condensates were administered intravenously in mice to determine the influence of peptide DNA formulation parameters on specific targeting to Kupffer cells. Optimal targeting to Kupffer cells required the combined use of 50 mol % Man9‐CWK18 and PEG‐CWK18 to mediate specific recognition by the mannose receptor to Kupffer cells. The cellular uptake of cross‐linked Man9‐CWK18/PEG‐CWK18 DNA co‐condensates was receptor mediated since Kupffer cell targeting was inhibited by pre‐administration of Man‐bovine serum albumin (BSA) but not BSA. An optimized formulation targeted 60% of the dose to the liver, with 80% of the liver‐targeted DNA localized to Kupffer cells. Cross‐linking with either 6, 15, or 50 mol equiv of glutaraldehyde led to a corresponding decrease in the metabolism rate of DNA in liver as measured by half‐live‐ of 4, 6, and 39 h, respectively. Tail vein dosing of 50 μg of DNA co‐condensates cross‐linked with 6 mol equiv of glutaraldehyde produced detectable levels of human α1‐antitrypsin in blood after 12 h, which peaked at day six and persisted for 10 days. The level of human α1‐antitrypsin was elevated two‐fold each day when dosing with DNA co‐condensates cross‐linked with 15 mol equiv of glutaraldehyde, revealing a correlation between the metabolic stability of the DNA in liver and level of gene expression. In addition to possessing greater metabolic stability, DNA co‐condensates cross‐linked with 50 mol equiv of glutaraldehyde, but lacking a targeting ligand, avoided rapid liver uptake and possessed a prolonged pharmacokinetic half‐life, providing insight into a means to target DNA condensates to peripheral tissues. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:2010–2022, 2001

Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions

An efficient Agrobacterium-mediated transformation procedure for barley was developed for immature embryos. Factors considered crucial for the delivery and integration of transgenes, such as type of growth regulator, duration of preculture period, plasmolysis and micro wounding were evaluated for their effect on frequencies of transient expression and stable integration. Dicamba in the callus induction and maintenance media was generally superior to 2,4-D in promoting transient expression and subsequent stable transformation. Plasmolysis generally reduced the level of transient expression although transgenic plants were recovered when 1-day precultured embryos were plasmolysed and grown on 2,4-D containing media. Micro wounding by particle bombardment did not change the level or localisation of transient expression events after Agrobacterium infection. Transgenic plants were generated with five out of 12 different procedures with transformation efficiencies ranging from 1.7 to 6.3%. Integration of bar and uidA coding sequences was confirmed by PCR for 75 plants representing 42 independent transgenic lines. Expression of the uidA gene was detected in 74% of primary transformants while 91% of them expressed the bar gene. Southern analysis proved integration of the transgenes with copy numbers ranging from one to six. Most transgenic lines that expressed both marker and reporter genes showed simple integration patterns and Mendelian inheritance of the transgenes in T1 progeny.

Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus-infected Sf9 cells.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1.5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5' upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3' end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.

Comparison of Constitutive Promoters for Sorghum [Sorghum bicolor (L.) Moench] Transformation

Sorghum [Sorghum bicolor (L.) Moench] transformation using microprojectile bombardment was achieved in 1993. Since then there have been very few additional reports on the topic. To develop a reliable method for foreign gene introduction, a promoter sequence capable of expression in the recipient cells is a critical requirement. Transient expression of the dual 35S cauliflower mosaic virus, rice actin-1, maize alcohol dehydrogenase-1, and maize ubiquitin-1 promoters driving the GUS (β-glucuronidase) gene in sorghum were compared. Transient β-glucuronidase activity in embryonic tissue was very low. It was concluded that there would be a significant benefit in investigating and isolating new constitutive promoter sequences potentially resulting in a higher level of transient expression in sorghum.

Transient expression of a reporter gene changes significantly during somatic embryogenesis in alfalfa

Transient expression of the β -glucuronidase (GUS) gene driven by a 35S constitutive promoter was investigated during somatic embryo development in alfalfa (Medicago sativa L.) following micro-projectile bombardment. The level of gene expression varied with the different developmental stages and changed dramatically within the stages. During embryogenic callus induction (stage I), expression was low in explants freshly transferred to culture, but increased when cells started to divide and show competence for somatic embryogenesis. Gene expression, however, decreased when extensive callus formation began and reached the lowest observed level when callus was fully developed. Reporter gene expression was consistently low during embryogenic cell proliferation (stage II). In contrast, high levels of reporter gene expression were detected in non-embryogenic suspension cultures during cell proliferation. Extremely low reporter gene expression was detected in embryonal tissues and young embryos at the embryo development and maturation stage (stage III). Expression increased as the somatic embryos developed and reached the highest level when the embryos reached maturity. Transient expression levels were similar in excised petiole tissue across 10 alfalfa genotypes, which included both diploid and tetraploid genotypes. In general, reporter gene expression was found to be higher in more organised mature tissues and organs than in less-organised or young tissues. The developmental status of the cells and tissues appears to be an important factor in the degree of transient expression of the introduced gene. Changes of gene expression during embryogenesis and factors affecting gene expression are discussed. Key words: GUS expression, Medicago sativa, bombardment, forage

High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells

We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommend to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.

Bean tissue culture and genetic transformation with Agrobacterium

In this paper we report the establishment methods of a rapidly growing callus culture of Phaseolus vulgaris bean as well as the conditions required for a high level of transient gene expression using Agrobacterium-mediated transformation. A vector is containing both the lindan-resistance gene as a selectable marker, and GUS gene as a screenable marker. By using hypocotyl explant and vertical culture on B5 medium supplemented with 1 mg/1 kinetin- and 2,4-D 2 mg/1 and subcultured every 3-4 weeks, we can recommend to get a good and much callus from bean. This will help in introducing foreign DNA into callus cells. One strain of Agrobacterium carrying plasmid as vector for introducing foreign DNA into plant cells was used. At different concentrations of lindan; 3, 4 and 4.5 mg/I, the transformed Maxidor callus survived and grew over a period of 6 month and subcultured every 3-4 weeks, but the control callus died. Callus were assayed for GUS activity to confirm the expression of the GUS gene using the histochemical assay test. The GUS gene was also correctly expressed in callus cultures grown on 4mg/I lindan-selected medium, the typical blue colour in the histochemical assay using the X-gluc as substrate. But the control, non-transformed callus was not able to grow in the presence of lindan, neither showed a positive reaction in the in vitro assays.

Retroviral promoter-trap insertion into a novel mammalian septin gene expressed during mouse neuronal development

We have characterized a retroviral promoter-trap insertion into a novel mammalian septin gene, Sep3. Its predicted amino acid sequence shares significant homology to that of Saccharomyces cerevisiae CDC3, CDC10, CDC11, CDC12, the Drosophila genes Pnut, Sep1, Sep2, and the mammalian genes BH5, CDC10, Nedd5, Diff6, and Sep2, which are implicated in cytokinesis and cell polarity. Sep3 encodes a protein of 465 amino acids, and contains an evolutionary conserved ATP/GTP-binding motif, two coiled-coil domains, and a highly hydrophobic domain at the C terminus. Alkaline phosphatase reporter gene expression in transgenic embryos was first detected at E8.5 in the neural fold, and high levels of expression continued throughout embryogenesis in the neural tube and brain. In addition, a low level of transient expression was detected in the somites, gut, and branchial arches of mouse embryos. Overall, reporter gene expression recapitulated Sep3 mRNA expression during mouse embryogenesis. In adults, Sep3 transcripts were only detected in the brain and testis. Zoo blot analysis revealed that Sep3-related sequences exist in several vertebrate species including zebrafish, frog, chicken, mouse and human. Consistent with the retroviral insertion into the 3′ UTR of the Sep3 gene, no obvious phenotypes associated with the promoter trap were detected in transgenic embryos or adult mice. In summary, we report the first isolation of a novel full-length Sep3 cDNA and extensive characterization of its expression during mouse embryogenesis and in adult tissues.

Physiological and genomic variations in rice cells recovered from direct immersion and storage in liquid nitrogen

The use of cryoprotectants and slow cooling rates are routine procedures for the cryopreservation of plant cell lines. However, our results with rice (Oryza sativa L., cv. Taipei 309) show that calli can be cryopreserved by direct immersion and stored in liquid nitrogen without any cryoprotection. The efficiency of recovery using this method, as well as a conventional method was generally increased with a previous abscisic acid (ABA) treatment. Following cryopreservation, calli demonstrated some differences with respect to unfrozen calli of the same lines. Thus, resistance to freezing stress (−20°C for 2 h) increased significantly in all lines tested, irrespective of their pre‐incubation with ABA. Calli that had been directly stored in liquid nitrogen also demonstrated a higher competence for genetic transformation than their unfrozen counterparts, as indicated by the transient gene expression levels obtained after particle bombardment. These differences might lead to further biotechnological applications. A genetic analysis of amplified DNA polymorphisms was performed with three independent lines that had been subjected to four combinations of ABA treatment and direct immersion in liquid nitrogen. At the loci screened with the randomly amplified polymorphic DNA (RAPD) markers tested, the genetic variations among lines and among calli of the same line akppear to be more related to tissue‐culture‐induced somaclonal variation than to cryoselection.

The pine Pschi4 promoter directs wound-induced transcription

Mechanical wounding stimulates the accumulation of Pschi4 transcripts (encoding a putative extracellular chitinase) in pine trees. To gain insight into the transcriptional regulatory region(s) in this gymnosperm defense gene, the 5′-flanking region of Pschi4 was fused to the uidA reporter gene encoding β-glucuronidase (GUS), and the construct was functionally tested for transient expression in onion and pine cells. As little as 200 bp of the upstream sequence was sufficient to promote activated transcription and nearly maximum levels of transient expression in onion cells. Similar results were obtained in bombarded pine cells, although maximum expression appeared to require the entire 4.5 kb of upstream sequence. Having defined potential regulatory regions in the 5′ flanking region of Pschi4 in transient assays, the pine promoter- uidA fusions were functionally tested in stably transformed tobacco plants. As little as 200 bp of the upstream sequence was sufficient to direct wound-inducible transcription. Furthermore, particle bombardment was sufficient to stimulate transgene expression in the transgenic tobacco, indicating that bombardment per se leads to wound responses in vivo. Taken as a whole, our data demonstrate that a gymnosperm promoter can recruit a wound-regulated signal transduction pathway in angiosperms. This suggests that the downstream components of wound signaling pathways are functionally conserved across broad taxonomic groups.

Transient gene expression in mammalian cells grown in serum-free suspension culture.

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 mug/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10-13:1. However, the ratio increases to 33:1 for 0.1-0.2 mug/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 mug/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2-5 l spinner culture scale within 3-5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.

Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea.

Gene Transferin vitroandin vivowith Epstein-Barr Virus-Based Episomal Vector Results in Markedly High Transient Expression in Rodent Cells

The EBV-based plasmid vectors, which carry oriP and EBNA1 gene from EBV genome, can be retained in the nucleus and replicate in human cells. Rodent cells are not permissive for the EBV plasmids, in terms of the plasmid replication. However, the EBV vector facilitates not only the long term maintenance of the plasmid but also high level gene expression at a transient phase after transfection. It has not been elucidated if rodent cells show this high level transient expression. We demonstrate that rodent cells transfected with an EBV vector expressed a marker gene more intensively than those with a conventional plasmid vector did. The high marker gene expression was also seen in rat myocardium injectedin vivowith the EBV plasmid. The present data indicate that the EBNA1-oriP system functions in “non-permissive” rodent cells at transient phase, and may require different cellular factor(s) for the transient expression and plasmid replication.

Efficient Gene Transfer to Human Endothelial Cells Using DNA Complexed to Adenovirus Particles

Human endothelial cells have been found to be relatively refractory to various methods of DNA transfection currently in common use. By using a transfection method involving DNA complexed with replication-deficient adenovirus particles, we have shown that 20% of a population of cultured endothelial cells can be transfected and high levels of transient expression achieved. Both early-passage human umbilical vein endothelial cells and the continuous differentiated line of human endothelium-derived EA.hy926 cells are responsive to this method of transfection. Efficient DNA transfection of endothelial cells is important for studies of endothelium-specific promoters and is a potentially useful route for transgenic therapy.

A Novel Method of Cell-Specific mRNA Transfection

In this study, we developed a cell-specific mRNA transfection system using streptavidin-protein A (ST-PA) fusion protein and monoclonal antibodies (mAbs). We previously reported that ST-PA fusion protein and mAb complexes can transfer certain biotinylated proteins into specific cell types. At this time, we combined anin vitrotranscribed biotinylated and self-replicating Sindbis virus genomic RNA with ST-PA fusion protein and mAbs. In the presence of cationic liposomes, to prevent RNA degradation, this complex is able to transfect a reporter gene to specific cancer cells in a mAb dose-dependent manner. Even in the absence of cationic liposomes, biotinylated mRNA, ST-PA fusion, and mAb complexes can transfer some types of cancer cell suspension cultures. This cell-specific transfection system is a novel method of introducing various mRNAs into cells that results in high levels of transient protein expression.

Cross-protection and selectable marker genes in plant transformation

Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks withAgrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration.

Using firefly luciferase to identify the transition from transient to stable expression in bombarded wheat scutellar tissue

Transformation of wheat by the biolistic procedure is unpredictable and inefficient. To gain insight into the fate of transgenes introduced into wheat scutellar tissue by particle bombardment, the expression of the firefly luciferase gene was followed using low‐light video imaging. Luciferase expression can be detected as early as 40 to 50 min after bombardment. After 48 h, the levels of transient expression in individual scutella fall rapidly, becoming undetectable 10 to 20 days later. Luciferase activity was observed to recover in a small percentage of the material and it was from this material that transformed plants, stably expressing luciferase, were recovered. We concluded that the calluses which recover luciferase expression are stably transformed and we have termed the transition period between transient and stable expression the transformation boundary. The percentage of scutella displaying luciferase activity, as measured at 30 days post‐bombardment, was judged a realistic measure of the efficiency of the transformation procedure. The results of our experiments suggest that the selection and regeneration of plants were not major factors contributing to the poor transformation efficiencies associated with biolistic transformation. The results demonstrate that luciferase can be used to assess rapidly and quantify the efficiency of the transformation procedure without the need to produce transformed plants. This will allow different procedures to be rapidly assessed and compared and should provide valuable insight into the conditions required to improve the efficiency of DNA integration and stable expression in species recalcitrant to transformation.

Study on transient expression of gus gene inChlorelia ellipsoidea (Chlorophyta) by using biolistic particle delivery system

Study on the transient expression of GUS gene at different growing stage ofChlorella ellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between microprojectile and target cell, bombardment times, are reported in this paper. The results showed thatC. ellipsoidea in exponential phase has higer level of transient expression and that treatment with osmosis can improve the GUS transient expression notably. The effect of distance or bombardment times was not observed.