Conditions For Transient Expression Research Articles

DnaJ molecules as potential effectors in Meloidogyne arenaria. An unexplored group of proteins in plant parasitic nematodes

ABSTRACTPlant pathogenic organisms secrete proteins called effectors that recognize, infect and promote disease within host cells. Bacteria, like Pseudomona syringae, use effectors with DnaJ function to disrupt plant defenses. DnaJ proteins (also called Hsp40) are a group of co-chaperone molecules, which assist in the folding of proteins. Despite the described role of DnaJs as effectors in several groups of pathogens, this group of proteins has never been correlated with the infection process in plant parasitic nematodes. In this study, we analyze the importance of DnaJ for plant parasitic nematodes. To do that, we compare the number of DnaJ proteins in nematodes with different lifestyles. Then, we predict the secreted DnaJ proteins in order to detect effector candidates. We found that Meloidogyne species have more secreted DnaJs than the rest of the nematodes analyzed in the study. Particularly, M. arenaria possess the highest proportion of secreted DnaJ sequences in comparison to total DnaJ proteins. Furthermore, we found in this species at least five sequences with a putative nuclear localization signal, three of them with a serine rich region with an unknown function. Then, we chose one of these sequences (MG599854) to perform an expression analysis. We found that MG599854 is over-expressed from 3 days post inoculation onwards in tomato plants. Moreover, MG599854 seems to be enough to produce cell death in Nicotiana benthamiana under transient expression conditions. In concordance with our results, we propose that DnaJ proteins are a potential source of effector proteins in plant parasitic nematodes.

OPTIMIZATION OF EXPRESSION CONDITIONS OF GENE ENCODING ANTIGEN M OF PRRSV IN LEAVES OF NICOTIANA BENTHAMIANA BY AGROINFILTRATION METHOD

Reproductive disorders and respiratory swine (Porcine Reproductive and Respiratory Syndrome - PRRS) is an infectious disease caused by the PRRS virus (PRRSV), spread fast speeds, causing mass death when infected pigs. M protein is one of the major structural protein of the PRRSV, has a molecular weight of about 19 kDa, encoded by the open reading frame 6 (ORF6), which is used to design a subunit vaccine against back PRRSV. In this study, conditions for transient expression of the gene encoded the M protein of PRRSV in leaves of the Nicotiana benthamiana by agroinfiltration method were determined. The results show that optimal conditions for transient expression of protein M in the leaf are used the simultaneous of the A. tumefaciens strain containing vector carrying the gene encoding the protein M and A. tumefaciens containing vector carrying gene encoding supported protein HC-Pro PVY, concentration of acetosyringone with 450 µM, the bacterial cell density used to infect into leaf with OD600 is 0.5, The physiological age of the leaf suitable for infecting bacteria was young leaf and mature leaf of tobacco plants 4 to 6 weeks of age, and the transient expression time of gene encoding protein M of PRRSV in tobacco leaves is most effective 6 days after infecting. This agroinfiltration method can be used to express large amounts of protein - antigen M of PRRSV in tobacco plants to produce vaccines against this virus.

Improving the quality of a recombinant rabbit monoclonal antibody against PLXDC2 by optimizing transient expression conditions and purification method

Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods. The quality and function of purified antibody preparations were checked by electrophoresis and western blot analysis. The secreted rabbit mAb produced by a combination of culturing at 32 °C, purification by ammonium sulfate fractionation, and diethylaminoethyl resin (DEAE) ion exchange chromatography was of high quality. In contrast, the antibody produced by the cells grown at 37 °C for 6 days after transfection and purified by Protein A/G affinity method was low quality. Hypothermic conditions during production reduced protein heterogeneity probably by favorably affecting the levels of glycosylation and aggregation. In particular, according to western blotting data, CIMmultus DEAE chromatography that utilizes monolithic columns not only excluded inferior charge variants resulting from nonspecific reactions but also yielded rabbit mAb that was of better quality than commercially available rabbit polyclonal antibodies. The combination of techniques suggested by us may be a general approach to enhance product quality of rabbit mAbs produced by transient expression systems.

Agroinfiltration of intact leaves as a method for the transient and stable transformation of saponin producing Maesa lanceolata

A method has been developed to genetically transform the medicinal plant Maesa lanceolata. Initially, we tested conditions for transient expression of GFP-bearing constructs in agroinfiltrated leaves. Leaf tissues of M. lanceolata were infiltrated with Agrobacterium tumefaciens carrying a nuclear-targeted GFP construct to allow the quantification of the transformation efficiency. The number of transfected cells was depended on the bacterial density, bacterial strains, the co-cultivation time, and presence of acetosyringone. The transient transformation assay generated the highest ratio of transfected cells over non-transfected cells upon 5 days post-infiltration using A. tumefaciens strain LBA4404 at an OD₆₀₀ = 1.0 in the presence of 100 μM acetosyringone and in the absence of a viral suppressor construct. In a second series of experiments we set up a stable transformation protocol that resulted in the regeneration of kanamycin-resistant plants expressing nuclear GFP. This transformation protocol will be used to introduce overexpression and RNAi constructs into M. lanceolata plants that may interfere with triterpenoid saponin biosynthesis. We have developed a transformation protocol for saponin producing Maesa lanceolata. Using the protocol reported here, now we are able to generate the tools for the modification of saponin production.

Transient GUS and GFP Expression in Spanish Red Cedar (<i>Cedrela odorata</i> L.) Somatic Embryos. Optimization of Bombardment Conditions and Evaluation of Selective Agent Lethal Dose

Cedrela odorata is a tropical tree widely appreciated for its wood. Commercial plantations are frequently hampered by the attack of the meliacea borer, Hypsipyla grandella, and the lack of resistant varieties. C. odorata traditional breeding would consume very long periods of time, thus direct transfer of entomotoxic coding genes to generate resistant varieties is a promising alternative. There are two prerequisites for gene manipulation of this species: 1) to set the conditions for transgene delivery and 2) to have a way to select regenerating transformed plants. In this paper, we report the optimal biolistics conditions for transient expression of uidA and gfp reporter genes in C. odorata somatic embryos and the selective doses for kanamycin, spectinomycin, phosphinotrycin and hygromycin to screen transformed cells.

Production of human interferon ALPHA 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

Transient Expression of Green Fluorescent Protein in Rice Calluses : Optimization of Parameters for Helios Gene Gun Device

An optimized condition for particle bombardment is necessary for efficient genetic transformation. Parameters for Helios gene gun, the new system for nucleic acid delivery which is mainly consists of hand-held device sold by Bio-Rad Laboratories (California USA), were examined based on transient expression of synthetic green fluorescent protein (sgfp) in rice calluses of indica cv. Fatmawati and japonica cv. Nipponbare. In the experimental conditions that we examined, parameters found to be the most favorable conditions for transient expression of sgfp in rice callus cells were as follows: 200−250 psi helium pressure, 0.6 μm gold particle size, 0.25 mg gold particles per shot, and 1.5 μg plasmid-DNA per shot. Desiccation of callus cells for eight min was also found appropriate. The level of transient sgfp expression was not significantly influenced by the pre-culture for 4 to 12 d before bombardment or by callus age between 10 and 33 wk old in Fatmawati. These parameters for this particular device should improve the transient expression, thus enabling stable expression of introduced genes via Helios gene gun using callus as a target tissue.

Effects of a range of beta2 adrenoceptor agonists on changes in intracellular cyclic AMP and on cyclic AMP driven gene expression in cultured human airway smooth muscle cells.

1. The effects of the selective beta2 adrenoceptor agonists salbutamol, terbutaline and salmeterol and the non-selective beta adrenoceptor agonist isoprenaline on [3H]-cyclic AMP formation and cyclic AMP response element (CRE) driven luciferase expression, assessed using the construct p6CRE/luc, were studied in primary cultures of human airway smooth muscle (HASM) cells. 2. Optimal transfection conditions for transient expression of pGL3 Control were 4 microg DNA/well71 in a 6 well plate and 1.8 microl Transfectam/microg DNA. Expression was maximal at 48 - 72 h. 3. Salbutamol (maximum response 19%, EC50 0.6 microM), terbutaline (maximum response 38%, EC50 2.3 microM) and salmeterol (maximum response 18%, EC50 0.0012 microM) were all partial agonists for cyclic AMP formation compared with isoprenaline (EC50 0.08 microM). However, all of the beta2 adrenoceptor agonists produced increases in CRE-driven luciferase activity, in cultured HASM transfected with the vector p6CRE/luc, which were equivalent or greater (salmeterol) than those seen with isoprenaline. 4. Both salbutamol and salmeterol were more potent at increasing luciferase expression than in elevating cyclic AMP levels in these cells. The potency ratios (EC50 (cyclic AMP)/EC50 (LUC)) for the agents studied were isoprenaline: 0. 2 fold, terbutaline: 3 fold, salbutamol: 24 fold, salmeterol: 38 fold. 5. These data suggest that important quantitative differences exist in the ability of beta2 adrenoceptor agonists to increase whole cell cyclic AMP levels in airway smooth muscle and to drive gene expression via a CRE-driven mechanism.

Optimization of transfection conditions for expression of green fluorescent protein in Drosophila melanogaster S2 cells

Drosophila melanogaster S2 cells provide attractive features for efficient expression of heterologous gene products. We report the optimization of transfection conditions for transient expression of green fluorescent protein (GFP) in Drosophila S2 cells by lipofectin and electroporation. GFP expression in lipofectin transfection was optimum at a transfection time of 24 h, a 5:1 ratio of lipofectin to DNA, and a seeding density of 4 × 10 5 cells/cm 2. On the other hand, GFP expression from electroporation transfection was optimum at 1125 V/cm, 250 μF, DNA content of 20 μg, and a seeding density of 1.0 × 10 7 cells/cuvette. Use of the optimum lipofectin method for transient expression resulted in an approximately 3-fold more efficient transfection than the electroporation method. We also investigated the stable transformation of Drosophila S2 cells by using the lipofectin method. Stably transformed polyclonal cell populations expressing GFP were isolated after 4 weeks of selection by using hygromycin B. The optimum ratio of plasmid pAc5CPPA-GFP to pCoHygro for the cotransfection of cells was 49 : 1 for GFP expression. Recombinant GFP expressed in stably transformed S2 cells was found primarily in the intracellular fraction with a molecular weight of 28 000. The maximum GFP concentration in the S2 cells was approximately 9 mg/l. We demonstrated the expression of GFP in Drosophila S2 cells and its use as a reporter for the analysis of gene expression.

Photosensitisation Properties of Mitochondrially Localised Green Fluorescent Protein

The photosensitisation properties of a mitochondrially localised green fluorescent protein (GFP) variant were established in cultured monkey kidney cells. We first cloned into a mammalian expression vector the thermostable variant GFP5, fused at its N-terminus to the 16-amino acid mitochondrial targeting sequence of human 3-oxoacyl-CoA thiolase. The recombinant plasmid thus constructed, pCZ34, was transfected into COS7 cells, under conditions for transient expression. GFP5 was shown to have a mitochondrial localisation by confocal microscopy, confirmed by its almost identical pattern with that of MitoTracker Red which selectively stains mitochondria. After photoirradiation of such transfected COS7 cells with 390-570 nm light to excite the mitochondrially localised GFP5, significant cell killing was observed. DAPI-staining of the dead cells detached from the growth support revealed cells to have undergone apoptosis. Thus, GFP5 can be used in the development of an organelle-specific photosensitiser since it can be targeted to different subcellular locations by protein engineering of the signal sequences.

Role of the A/B Region of the Human Mineralocorticoid Receptor in Aldosterone Response Selectivity

In transfection experiments performed with wild-type MR, the maximal transcriptional activation of a mineralocorticoid response induced by glucocorticoid was generally similar to that of aldosterone, the natural mineralocorticoid hormone. However, compared to aldosterone, glucocorticoid activity decreased when the A/B region of MR was absent. We describe in this study the synthesis and biological activities of seven mutated MRs differently truncated in the N-terminal region. Using transient expression conditions in MCF-7 cells, the N-terminal domain of MR has been shown to contain a region (residues 254-390) whose deletion led to an apparent "aldosterone selectivity". These results suggest that this region could help to maintain the most transcriptionally active conformation of MR even in the presence of ligands which are not by themselves able to fully induce such a conformation.

Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation.

The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.

Dominant negative regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain and heterodimer formation.

A series of deletion mutants was constructed for the rat androgen receptor (AR) to delineate sequences involved in transcriptional activation. Using transient expression conditions in CV-1 cells and in vitro DNA-binding studies, the amino-terminal domain of the receptor was shown to contain a region (residues 147-296) that is mandatory for trans-activation. Receptors with deletions (residues 147-408) in the N-terminal domain, but with intact DNA- and ligand-binding domains, interacted in vitro with androgen-responsive elements albeit with affinities lower than that of the wild type receptor. Coexpression of N-terminal deletion mutants (delta 46-408 and delta 38-296) with the wild type AR blunted trans-activation by the latter protein in a dominant fashion. By contrast, a hormone-binding-deficient receptor (delta 788-902) that had poor intrinsic activity potentiated the trans-activation by the native receptor. Mechanisms by which deletion mutants in the N-terminal region abolish the function of the wild type protein appear to involve heterodimer formation during interaction with DNA and direct competition for available binding sites on DNA, rather than squelching of accessory proteins. In contrast to impaired trans-activation, binding of the ligand to N-terminal deletion mutants brought about conformational changes that were comparable in wild type and mutant forms, as judged by electrophoretic mobility shift assays. Taken together, these data have specified a region in the N-terminal domain of the AR that plays a decisive role in transcriptional regulation.

Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures

Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system.

A correlation between dexamethasone inducibility and basal expression levels of retroviral vector proviruses.

Identical transcription units inserted at different positions of mammalian chromosomes may vary widely in transcriptional activity. We have used a set of ten cell clones with random unselected single integrations of retroviral vectors to study such position effects. The vector used carries a neo gene driven by the Akv murine leukemia virus long terminal repeat that has only a weak promoter-enhancer activity in the target cell, the lymphoid cell line L691. Under transient expression conditions, the strength of the Akv promoter-enhancer in the L691 cells is increased by dexamethasone. In cell clones with single vector integrations, a correlation is observed between the non-induced expression levels and the degree of dexamethasone induction. The strongest relative induction is found for the integrated vectors with the lowest non-induced expression levels and approaches the inducibility under transient expression. These results indicate that expression levels are composed of distinct contributions from the integrated vector and from the site of integration and are best explained in terms of a model in which the sites of chromosomal integration exert variable positive enhancer effects upon vector transcription.

Human immunodeficiency virus type 1 (HIV-1) provirus expression and LTR transcription are repressed in NEF-expressing cell lines

Human immunodeficiency virus type 1 (HIV-1) NEF protein has been demonstrated to be a negative regulator of HIV-1 replication and HIV-1 LTR transcription under transient expression conditions. The difficulty of several laboratories to reproduce these findings led us to reexamine the role of NEF in HIV-1 provirus expression and HIV-1 LTR transcription. Basal transcription from the HIV-1 LTR in the presence of a NEF expression vector was compared to that in the presence of a mutated NEF vector. NEF expression led to a greater than 10-fold repression of LTR transcription under these conditions. HeLa and Jurkat cell lines carrying the nef gene linked to the CMV promoter or the HIV-1 LTR were isolated by coselection for neomycin resistance. Single cell isolates were further selected for the expression of nef transcripts. With the exception of the anti-sense nef cell lines, all the nef cell lines expressed the 27-kDa NEF protein, detectable by immunoprecipitation. NEF + HeLa cell lines were at least 5-fold less efficient than NEF − HeLa cell lines in transient proviral expression. Provirus expression was also repressed in the NEF + Jurkat cell lines. TAT-activated LTR transcription from an HIV-1 LTR-linked CAT expression vector was repressed 10-fold in the NEF + HeLa and NEF + Jurkat cell lines. When infected with HIV-1, NEF expressing T lymphoid cell lines showed moderate delays in onset and peak of reverse transcriptase production. However, none of these cell lines completely arrested virus replication. Our data confirm a negative regulatory effect of NEF on both virus production and LTR driven CAT expression in the cell lines tested. It is possible that cell specific factors may influence NEF activity.

Genotype- and promoter-induced variability in transient ?-glucuronidase expression in pea protoplasts

Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of β-glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 μF; and using 125 μg of calf thymus carrier DNA and 75 μ of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.

Identification of sequences in the long terminal repeat of avian sarcoma virus required for efficient transcription

Two different vectors, LTR-NEO-LTR and LTR-CAT-LTR, were constructed and deletions were introduced in the upstream LTR at −299 and at −140. These deletion mutants were introduced into QT6 cells under transient expression conditions, and the levels of transcription were monitored by dot blot hybridization or by CAT assays. The results indicate that the nucleotides between −208 and −201 and between −141 and −119 in the U3 region of RSV LTR are required for efficient transcription.

Establishment of a transient expression system forDictyostelium discoideum

We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes.