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PubMedDate not supplied

Comparison of transient protein expression in tobacco leaves and plant suspension culture.

Transient gene expression is being developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor-intensive and time-consuming process of generating stably transformed transgenic plants. Transient expression of the gus-intron reporter gene was facilitated in three different tobacco species. Two different approaches to T-DNA delivery were compared: (1) infiltration of a prototrophic strain of Agrobacterium into leaves and (2) coculture of plant cell suspension cultures with an Agrobacterium auxotroph. Wounding of plant tissues with a wire brush prior to infiltration had a large positive impact on Nicotianabenthamiana leaves but not for Nicotiana tabacum or Nicotiana glutinosa. The best expression level achieved by leaf infiltration was in N. benthamiana (0.025% total soluble protein). A cell suspension culture line of N. glutinosa achieved an expression level greater than 0.04% TSP. The tissue culture-based technique therefore provides improved levels of transient expression under aseptic conditions to facilitate improvements in expression by control of the plant cell culture and Agrobacterium coculture environments.

Cells, Cultured↗

PubMed1989

Transient gene expression in aleurone protoplasts isolated from developing caryopses of barley and wheat.

Methods have been developed for the isolation of aleurone protoplasts from developing caryopses of Hordeum vulgare and Triticum aestivum in order to study transient expression of introduced genes. Chimaeric gene constructs were introduced into aleurone protoplasts by polyethylene glycol (PEG). Transient expression directed by the 35S promoter from cauliflower mosaic virus (CaMV) of the reporter gene encoding chloramphenicol acetyl transferase (CAT) was detected in aleurone protoplasts from developing barley and wheat grains. Using a similar construct, CAT activity increased when the alcohol dehydrogenase intron 1 fragment from maize was ligated between the 35S promoter and the CAT coding region. The demonstration of transient expression in protoplasts from developing aleurone layers indicates that they may be useful for investigating tissue and developmental control of genes coding for cereal seed proteins.

Brassica↗

PubMed1999

Variation of transient gene expression within single lineages of Trypanosoma cruzi.

The biodiversity of Trypanosoma cruzi is one of the main factors complicating the understanding of its molecular epidemiology. As an alternative to classical genetic methods, investigators have used DNA-mediated transformation techniques to study this diversity. Recently, transient expression data were shown to correlate with the genetic data. This led investigators to speculate on a potential speciation within clonal populations of Trypanosoma cruzi. To further test the phylogenetic significance of transient expression analysis in Trypanosoma cruzi, multiple plasmids were used to drive the expression of a reporter gene in different parasite populations. In our study, population specific expression of the reporter gene was observed but the variability revealed by these transient expression assays was very large and did not follow the grouping of Trypanosoma cruzi populations in 2 lineages. In this report, we discuss some of the limitations of transient assays on such a diverse parasite.

Animals↗

PubMed1996

A possible role of transient AChE expression in the medial geniculate of developing rats.

Acetylcholinesterase (AChE) and calbindin D-28K (CaBP) are transiently expressed in the rat auditory nuclei during the early postnatal period. In the ventral division of the medial geniculate body (MGV), the transient AChE expression in the neuropil is replaced by CaBP expression in the neurones. The time correlation between the up- and down-regulations in these neuro-chemicals suggests some switching over mechanism. A lesion of the inferior colliculus (IC) decreases the AChE reactivity in terminal field of the IC-MGV projections. We here demonstrate that the IC lesion results in CaBP expressions in neurones of the MGV before its normal onset. It is thus possible that the transiently expressed AChE plays an important role in the intercellular signal transduction for neurochemical phenotype expressions.

Acetylcholinesterase↗

PubMed2005

Optimisation and evaluation of a high-throughput mammalian protein expression system.

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.

Animals↗

PubMed2006

Transient gene expression by nonintegrating lentiviral vectors.

Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.

Antigens, CD34↗

PubMed1986

Human growth hormone as a reporter gene in regulation studies employing transient gene expression.

The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.

Animals↗

PubMed1991

Functional analysis of the pathogenesis-related 1a protein gene minimal promoter region. Comparison of reporter gene expression in transient and in stable transfections.

Pathogenesis-related (PR) proteins are a heterogeneous group of host encoded, low-molecular-mass proteins that are induced in plants by various external stimuli, such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5'-flanking region of the tobacco PR-1a gene [Pfitzner U.M., Pfitzner, A.J.P. & Goodman, H.M. (1988) Mol. Gen. Genet. 211, 290-295] was joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. Expression of the reporter gene was monitored in transient expression assays as well as in stable transformants. The PR-1a 5'-flanking sequences from -335, -149 or -71 to +28 are functional promoter elements in tobacco and carrot protoplasts, as determined by transient expression. These constructs direct correct initiation at the normal transcription-start site of the PR-1a gene. The level of gene expression was about twofold less than that obtained with the cauliflower mosaic virus 35S RNA promoter. Regulation of gene expression by acetylsalicylic acid, however, could not be detected in the transient assays. When the same constructs were stably integrated into the tobacco genome, neither constitutive nor induced beta-glucuronidase activity was observed. A comparison of the results from the transient and the stable transfection experiments suggests that expression of the reporter gene may be due to a constitutive transcriptional activity of the PR-1a 5'-flanking regions under the conditions of the transient assays and that the PR-1a promoter may contain at least two functional domains.

Aspirin↗

PubMed1999

Signal transduction in atria and ventricles of mice with transient cardiac expression of activated G protein alpha(q).

We recently showed that the transient expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the G protein alpha(q) subunit (HAalpha(q)*) in the hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after HAalpha(q)* protein becomes undetectable. We demonstrated that the activity of phospholipase Cbeta, the immediate downstream target of activated Galpha(q), is increased at 2 weeks, when HAalpha(q)* is expressed, but also at 10 weeks, when HAalpha(q)* is no longer detectable. This observation suggested that the transient HAalpha(q)* expression causes multiple, persistent changes in cellular signaling pathways. We now demonstrate changes in the level, activity, or both of several signaling components, including changes in the amount and hormone responsiveness of phospholipase Cbeta enzymes, in the basal level of diacylglycerol (which predominantly reflects activation of phospholipase D), in the amount or distribution of protein kinase C (PKC) isoforms (PKCalpha, PKCdelta, and PKCepsilon), and in the amount of several endogenous G proteins. These changes vary depending on the isoform of the signaling molecule, the chamber in which it is expressed, and the presence or absence of HAalpha(q)*. Our results suggest that a network of linked signaling functions determines the development of hypertrophy. They also suggest that atria and ventricles represent different signaling domains. It is likely that such changes occur in other model systems in which the activity of a single signaling component is increased, either due to an activating mutation or due to overexpression of the wild type.

Animals↗

PubMed1996

Ubiquitin gene expression following transient forebrain ischemia.

Ubiquitin gene expression following transient forebrain ischemia in the rat was analyzed by three probes which were specific for UbC, UbB and UbS30 mRNA. According to the in situ hybridization studies, each type of ubiquitin gene expression decreased at 30 min of reperfusion following 20 min of forebrain ischemia, thereafter increased, and then reached a peak at 4-6 h, both in the cortex and hippocampus. These changes returned to the control level after 24-48 h of recirculation. Among the three ubiquitin transcripts, changes in UbC expression were more marked in the hippocampus, and persistent expression of UbC transcripts in the CA1 and CA3 regions was observed at 24 h of reperfusion. With dot-blot analysis, significant increases in the UbC transcripts were noted at 4 h of reperfusion in the hippocampus, and at 6 h in the cortex following 20 min of ischemia. These results suggest that changes in UbC expression might be a good indicator of ischemic stress.

Animals↗

PubMed1994

Structural organization of the human tyrosinase gene and sequence analysis and characterization of its promoter region.

Tyrosinase is the principal enzyme in the biosynthesis of melanin. The expression of tyrosinase is tissue-specific and appears to be regulated by various hormonal and environmental factors. Elucidation of the genomic structure and molecular basis of control of tyrosinase gene expression will greatly enhance our understanding of the regulation of human pigmentation. To this end, we have isolated and performed restriction mapping of recombinant cosmid and lambda phage clones containing the human tyrosinase gene, sequenced a 2.2-kilobase (kb) region of its promoter, and determined the potential regions regulating the tyrosinase gene expression in transient-expression system. The human tyrosinase gene is comprised of five exons and four introns. Based on our restriction mapping studies, the gene spans a distance of over 65-kb on chromosome 11 (q14-->q21). We constructed a series of plasmids (pHTY-CAT) that contain 5' sequential deletions of the human tyrosinase 5' flanking sequence fused to the reporter gene, chloramphenicol acetyltransferase (CAT). The plasmids were used to locate promoter regions that are potential regulators of tyrosinase gene expression in a transient expression system using melanoma cell lines. In human melanoma cells, the plasmid construct with a -2020 base pair (bp) promoter yielded the highest CAT activity. When the deletions reached -1739 bp, the CAT activity was dramatically reduced, indicating that important enhancer elements for transcription control are present between -1739 and -2020 bp. Further deletions up to -550 bp also resulted in dramatic decreases of CAT activity. However, when the deletion included -550 bp of the 5' flanking sequence, there was 26 percent of the CAT activity compared to that of the -2020 bp promoter. Deletions beyond -550 bp also showed markedly decreased CAT activity. Based on our data, we suggest that human tyrosinase gene expression is governed by both tissue-specific and multiple regulatory elements.

Base Sequence↗

PubMed1985

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.

Acetyltransferases↗

PubMed1990

Identification of the base-pair substitution responsible for a human acid alpha glucosidase allele with lower "affinity" for glycogen (GAA 2) and transient gene expression in deficient cells.

The lysosomal enzyme termed acid alpha glucosidase (GAA), or acid maltase, is genetically polymorphic, with three alleles segregating in the normal population. The rarer GAA 2 allozyme has a lower affinity for glycogen and starch but not for lower-molecular-weight substrates. The GAA 2 allozyme can be detected by "affinity" electrophoresis in starch gel, since the lower affinity for the starch matrix results in a more rapid migration to the anode. Previously, we have isolated and sequenced the cDNA for GAA and transiently expressed the cDNA in deficient fibroblasts. In order to determine the molecular basis for the GAA 2 allozyme, we constructed a cDNA and a genomic DNA library from a GAA 2 cell line and determined the nucleotide sequence of the coding region. Only a single base-pair substitution of an A for a G at base-pair 271 was found, resulting in substitution of asparagine for aspartic acid at codon 91. This amino acid substitution is consistent with the more basic pI of the GAA 2 enzyme. The base-pair substitution also abolishes a Taq-I site, predicting the generation of a larger DNA fragment. This larger Taq-I fragment was also seen in two other individuals expressing the GAA 2 allozyme. A 5' fragment containing the base-pair substitution was ligated to the remaining 3' cDNA from a GAA 1 allele and cloned into an expression vector, and the hybrid cDNA was transiently expressed in SV40-transformed GAA-deficient fibroblasts. The enzyme activity exhibited the altered mobility of the GAA 2 allozyme, as demonstrated by electrophoresis in starch gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1991

Transient gene expression in cassava using high-velocity microprojectiles.

The bacterial gene encoding beta-glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter from Arabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3' ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.

Cloning, Molecular↗

PubMed2003

Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells.

One of the major constraints to performing large-scale transfections of cultured mammalian cells for the transient expression of recombinant proteins is the production of large quantities of purified plasmid DNA. In this report partially purified plasmid DNA was prepared by a method that combines alkaline lysis of E. coli with standard precipitation techniques. The efficiency of calcium phosphate-DNA co-precipitate formation with crude DNA was similar to that observed for pure DNA, but precipitate formed with crude DNA also contained RNA. The transfection of adherent and suspension-adapted HEK293-EBNA cells with partially purified pEGFPN1 resulted in levels of transient GFP expression equivalent to those achieved with pure DNA. In addition, the co-transfection of 1-200 ml cultures of suspension-adapted HEK293-EBNA cells with two different plasmids encoding the heavy and light chain genes of anti-human RhD IgG1, respectively, yielded similar IgG titers with pure and partially purified plasmid DNA. Finally, it was observed that suspension-adapted cells were more tolerant to the presence of RNA in the plasmid preparations than were adherent cells. These findings are relevant to the field of DNA transfection, including applications ranging from high-throughput screening to large-scale transient protein expression.

Bioreactors↗

PubMed1993

Transient gene expression by SV40 promoter characterizes sequential differentiation of embryonal carcinoma F9 cells into primitive and visceral endoderm.

Transient expression of the chloramphenicol acetyltransferase (CAT) gene under the control of simian virus 40 (SV40)y Moloney murine leukemia virus, human T cell leukemia virus, and cytomegalovirus promoters was stimulated by the differentiation of F9 stem cells into primitive endoderm, but repressed again by further differentiation into visceral endoderm. Deletion mutants of the SV40 enhancer showed that a similar set of motifs is critical for CAT expression at all stages of F9 differentiation, but differentiation dependency was observed even in their absence. The stability of transient gene expression under the control of the SV40 promoter was markedly dependent on F9 differentiation. Appreciable expression was detected even in undifferentiated F9 cells immediately after gene transfection, was maximal at 12 h and declined rapidly thereafter. On the other hand, expression in primitive endoderm increased until 72 h. The decline was accelerated again in visceral endoderm. This shift was somewhat specific to the virus promoter since CAT expression in undifferentiated F9 cells under the control of the elongation factor 1 alpha promoter was more stable than for virus promoters tested. Thus, the change in stability of expression is important for differentiation-dependent virus promoter activity.

Animals↗

PubMed1990

Transient gene expression in maize, rice, and wheat cells using an airgun apparatus.

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The beta-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 x 10(-3). In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals.

Journal Article↗

PubMedDate not supplied

Concomitant cortical expression of TNF-alpha and IL-1 beta mRNAs follows early response gene expression in transient focal ischemia.

The expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) mRNAs was significantly increased in the rat ischemic cortex following temporary occlusion of the middle cerebral artery (TMCAO) with reperfusion. Northern blot analysis demonstrated that the induction of TNF-alpha and IL-1 beta mRNAs occurred as early as 1 h after reperfusion, exhibiting a 4.6-fold increase (p < 0.05, n = 4) and 6.8-fold increase (p < 0.05, n = 4) in the ischemic cortex over control, respectively. TNF-alpha mRNA reached its peak at 3 h (8.0-fold, p < 0.05), whereas IL-1 beta mRNA reached its peak at 6 h (29.5-fold, p < 0.05). Both cytokine mRNA levels remained elevated for up to 2 d after reperfusion. In contrast to the time course of these cytokine mRNAs, c-fos and zif268 mRNAs, two early response genes, displayed a greater and earlier time-response profile. The early induction of c-fos and zif268 mRNAs in temporary brain ischemia with reperfusion suggests their roles in transcriptional regulation. The later concomitant expression of TNF-alpha and IL-1 beta suggests that these cytokines play an important role in the inflammatory response associated with focal ischemia.

Animals↗