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1.  Comparison of Expression Vectors for Transient Expression of Recombinant Proteins in Plants 
Production of recombinant proteins in plants is of increasing importance for practical applications. However, the production of stable transformed transgenic plants is a lengthy procedure. Transient expression, on the other hand, can deliver recombinant proteins within a week, and many viral vectors have been constructed for that purpose. Each of them is reported to be highly efficient, robust and cost-effective. Here, a variety of expression vectors which were designed for transient and stable plant transformation, including pPZP3425, pPZP5025, pPZPTRBO, pJLTRBO, pEAQ-HT and pBY030-2R, was compared for the expression of green fluorescent protein and β-glucuronidase in Nicotiana benthamiana by Agrobacterium-mediated transient expression. Our results show that pPZPTRBO, pJLTRBO and pEAQ-HT had comparable expression levels without co-infiltration of a RNA-silencing inhibitor. The other vectors, including the non-viral vectors pPZP5025 and pPZP3425, needed co-infiltration of the RNA-silencing inhibitor P19 to give good expression levels.
Electronic supplementary material
The online version of this article (doi:10.1007/s11105-013-0614-z) contains supplementary material, which is available to authorized users.
PMCID: PMC3881577  PMID: 24415845
Transient expression; Recombinant protein; Agroinfiltration; Viral vector; pPZP vector family
2.  Transformation of Rhizobia with Broad-Host-Range Plasmids by Using a Freeze-Thaw Method 
Several species of rhizobia were successfully transformed with broad-host-range plasmids of different replicons by using a modified freeze-thaw method. A generic binary vector (pPZP211) was maintained in Mesorhizobium loti without selection and stably inherited during nodulation. The method could extend the potential of rhizobia as a vehicle for plant transformation.
PMCID: PMC1393188  PMID: 16517691
3.  A Human Anti-Pseudomonas aeruginosa Serotype O6ad Immunoglobulin G1 Expressed in Transgenic Tobacco Is Capable of Recruiting Immune System Effector Function In Vitro▿  
The production of a recombinant human IgG1 in transgenic tobacco was examined to determine whether a plant-derived antibody could recruit immune system effector function against a bacterial pathogen. A plant transformation vector was engineered to contain genes for a human kappa light chain and a human gamma-1 heavy chain with VH and VL sequences from a previously identified human IgG2 monoclonal antibody (MAb) that specifically binds to and opsonizes Pseudomonas aeruginosa serotype O6ad. Unique NcoI and NotI restriction sites were incorporated to flank these variable sequences, resulting in a plant transformation vector that could be engineered for expression of any other human IgG1 antibody, requiring only the substitution of other VH and VL antigen-binding coding sequences. The plant-produced IgG1 was determined to have high-mannose glycan content and to be capable of mediating opsonophagocytosis of P. aeruginosa serotype O6ad in vitro using human complement and human polymorphonuclear leukocytes. Thus, MAbs produced in plants from this vector could provide human IgG1 MAbs for targeting other pathogens that require the recruitment of immune system effector functions.
PMCID: PMC2043195  PMID: 17606688
4.  The promoter of a plant defensin gene directs specific expression in nematode-induced syncytia in Arabidopsis roots 
Plant Physiology and Biochemistry  2011;49(10):1100-1107.
The beet cyst nematode Heterodera schachtii induces a feeding site, called syncytium, in roots of host plants. In Arabidopsis, one of the genes whose expression is strongly induced in these structures is Pdf2.1 which codes for an antimicrobial plant defensin. Arabidopsis has 13 plant defensin genes. Besides Pdf2.1, the Pdf2.2 and Pdf2.3 genes were strongly expressed in syncytia and therefore the expression of all three Pdf genes was studied in detail. The promoter of the Pdf2.1 gene turned out to be an interesting candidate to drive a syncytium-specific expression of foreign genes as RT-PCR showed that apart from the feeding site it was only expressed in siliques (seeds). The Pdf2.2 and Pdf2.3 genes were in addition expressed in seedlings, roots, leaves, stems, and flowers. These results were supported by the analysis of promoter::GUS lines. After infection with H. schachtii all GUS lines showed a strong staining in syncytia at 5 and 15 dpi. This expression pattern was confirmed by in situ RT-PCR.
► 3 plant defensin genes are strongly expressed in syncytia. ► Pdf2.1 is expressed in syncytia and seeds. ► The Pdf2.1 promoter is useful for the expression of foreign genes in syncytia.
PMCID: PMC3185291  PMID: 21813283
Plant defensin; Antimicrobial peptide; Syncytium; Heterodera schachtii; Arabidopsis; Roots; Dpi, days post infection
5.  Strict De Novo Methylation of the 35S Enhancer Sequence in Gentian 
PLoS ONE  2010;5(3):e9670.
A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora × G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (−90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (−148 to −85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the −149 to −124 and −107 to −83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants.
PMCID: PMC2843634  PMID: 20351783
6.  Molecular Cloning, Nucleotide Sequence, and Expression in Escherichia coli of a Hemolytic Toxin (Aerolysin) Gene from Aeromonas trota 
Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn5Km1 to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb NotI fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn5Km1. The 8.7-kb NotI DNA fragment was cloned into the vector pGEM5Zf(−) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in Escherichia coli JM109. The nucleotide sequence of the aerA gene, located on the 1.8-kb ApaI-EcoRI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon. An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the aerA gene codes for a 54-kDa protein, in agreement with nucleotide sequence data. The deduced amino acid sequence of the aerA gene product of A. trota exhibited 99% homology with the amino acid sequence of the aerA product of Aeromonas sobria AB3 and 57% homology with the amino acid sequences of the products of the aerA genes of Aeromonas salmonicida 17-2 and A. sobria 33.
PMCID: PMC106413  PMID: 9647817
7.  Structure-Based Engineering Increased the Catalytic Turnover Rate of a Novel Phenazine Prenyltransferase 
PLoS ONE  2012;7(10):e48427.
Prenyltransferases (PTs) catalyze the regioselective transfer of prenyl moieties onto aromatic substrates in biosynthetic pathways of microbial secondary metabolites. Therefore, these enzymes contribute to the chemical diversity of natural products. Prenylation is frequently essential for the pharmacological properties of these metabolites, including their antibiotic and antitumor activities. Recently, the first phenazine PTs, termed EpzP and PpzP, were isolated and biochemically characterized. The two enzymes play a central role in the biosynthesis of endophenazines by catalyzing the regiospecific prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dhPCA) in the secondary metabolism of two different Streptomyces strains. Here we report crystal structures of EpzP in its unliganded state as well as bound to S-thiolodiphosphate (SPP), thus defining the first three-dimensional structures for any phenazine PT. A model of a ternary complex resulted from in silico modeling of dhPCA and site-directed mutagenesis. The structural analysis provides detailed insight into the likely mechanism of phenazine prenylation. The catalytic mechanism suggested by the structure identifies amino acids that are required for catalysis. Inspection of the structures and the model of the ternary complex furthermore allowed us to rationally engineer EpzP variants with up to 14-fold higher catalytic reaction rate compared to the wild-type enzyme. This study therefore provides a solid foundation for additional enzyme modifications that should result in efficient, tailor-made biocatalysts for phenazines production.
PMCID: PMC3485228  PMID: 23119011
8.  A doxycycline-inducible urokinase receptor (uPAR) upregulates uPAR activities including resistance to anoikis in human prostate cancer cell lines 
Molecular Cancer  2007;6:34.
The urokinase receptor (uPAR) mediates a diverse array of cellular processes including several events involved in prostate cancer metastasis. Many of these activities are initiated or enhanced by uPAR binding to its proteolytic ligand, urokinase (uPA). Our objective in this study was to generate and test an inducible lentiviral system capable of expressing uPAR and DsRed fluorescent protein in human prostate cancer cell lines.
A DsRed-uPAR fusion construct was inserted into a lentiviral vector. Transduction of human prostate cancer cell lines with this virus and with a virus containing a reverse-tetracycline transactivator (rt-TA) resulted in a stable transgene which induced both uPAR and DsRed proteins in a dose-responsive fashion upon stimulation with doxycycline. Immunoblots and immunofluorescence studies indicated no detectable uPAR expression in non-induced prostate cancer cell lines. Cells with induced-uPAR demonstrated increased cellular adhesion to the matrix substrate vitronectin and increased net cell proliferation compared to uninduced cells. Finally, induced uPAR-expressing prostate cancer cells were resistant to anoikis over an extended time period when grown in suspension.
This doxycycline-inducible lentivirus system produces titerable levels of biologically active uPAR in vitro. This tool can be used to dissect cellular events following induction of uPAR in prostate cancer cells.
PMCID: PMC1885813  PMID: 17509140
9.  One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE 
PLoS ONE  2012;7(10):e47885.
Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.
PMCID: PMC3480454  PMID: 23112864
10.  Immunocontraception in Wild Horses (Equus caballus) Extends Reproductive Cycling Beyond the Normal Breeding Season 
PLoS ONE  2010;5(10):e13635.
Although the physiological effects of immunocontraceptive treatment with porcine zona pellucida (PZP) have been well studied, little is known about PZP's effects on the scheduling of reproductive cycling. Recent behavioral research has suggested that recipients of PZP extend the receptive breeding period into what is normally the non-breeding season.
Methodology/Principal Findings
To determine if this is the case, we compiled foaling data from wild horses (Equus caballus) living on Shackleford Banks, North Carolina for 4 years pre- and 8 years post-contraception management with PZP (pre-contraception, n = 65 births from 45 mares; post-contraception, n = 97 births from 46 mares). Gestation lasts approximately 11–12 months in wild horses, placing conception at approximately 11.5 months prior to birth. Since the contraception program began in January 2000, foaling has occurred over a significantly broader range than it had before the contraception program. Foaling in PZP recipients (n = 45 births from 27 mares) has consistently occurred over a broader range than has foaling in non-recipients (n = 52 births from 19 mares). In addition, current recipients of PZP foaled later in the year than did prior recipient and non-recipient mares. Females receiving more consecutive PZP applications gave birth later in the season than did females receiving fewer applications. Finally, the efficacy of PZP declined with increasing consecutive applications before reaching 100% after five consecutive applications.
For a gregarious species such as the horse, the extension of reproductive cycling into the fall months has important social consequences, including decreased group stability and the extension of male reproductive behavior. In addition, reproductive cycling into the fall months could have long-term effects on foal survivorship. Managers should consider these factors before enacting immunocontraceptive programs in new populations. We suggest minor alterations to management strategies to help alleviate such unintended effects in new populations.
PMCID: PMC2964306  PMID: 21049017
11.  Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. 
Journal of Bacteriology  1990;172(11):6568-6572.
A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.
PMCID: PMC526846  PMID: 2172217
12.  Identification and characterization of an Epstein-Barr virus early antigen that is encoded by the NotI repeats. 
Journal of Virology  1989;63(11):4609-4615.
The Epstein-Barr virus (EBV) genome is characterized by two regions carrying partially homologous clusters of short tandem repeats (NotI and PstI repeats) flanked by 1,044 and 1,045 base pairs with almost perfect homology (DL and DR, left and right duplications, respectively). Both repetitive regions are transcribed into poly(A)+ mRNA after induction of the productive EBV cycle with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and contain open reading frames. To identify the potential protein encoded by the NotI repeat open reading frame (BHLF1), two repeat units of EBV strain M-ABA were expressed using the tryptophan-regulated Escherichia coli expression vector pATH11. Rabbit antisera generated against the resulting fusion protein reacted specifically with a protein varying in molecular size between 70,000 and 90,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, found after 12-O-tetradecanoyl-phorbol-13-acetate or n-butyrate induction in various cell lines harboring EBV. In immunofluorescence tests with the BHLF1-specific antiserum, an immunofluorescence with EA-D specificity could be observed. In addition, the BHLF1 protein is exhibiting polyanion-binding activity with a maximum for single-stranded DNA. Furthermore, the fusion protein is recognized by a number of human EBV-positive sera.
PMCID: PMC251094  PMID: 2552144
13.  Monitoring of Gene Expression in Bacteria during Infections Using an Adaptable Set of Bioluminescent, Fluorescent and Colorigenic Fusion Vectors 
PLoS ONE  2011;6(6):e20425.
A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfpmut3.1, amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.
PMCID: PMC3108616  PMID: 21673990
14.  β-endorphin expression in the mouse retina 
The Journal of comparative neurology  2010;518(15):3130-3148.
Evidence showing expression of endogenous opioids in the mammalian retina is sparse. In the present study we examined a transgenic mouse line expressing an obligate dimerized form of Discosoma Red Fluorescent Protein (DsRed) under the control of the pro-opiomelanocortin promoter and distal upstream regulatory elements to assess whether pro-opiomelanocortin peptide (POMC), and its opioid cleavage product, β-endorphin, are expressed in the mouse retina. Using double label immunohistochemistry, we found that DsRed fluorescence was restricted to a subset of GAD-67-positive cholinergic amacrine cells of both orthotopic and displaced subtypes. About 50% of cholinergic amacrine cells colocalized DsRed and a large fraction of DsRed-expressing amacrine cells was positive for β-endorphin immunostaining, whereas β-endorphin immunoreactive neurons were absent in retinas of POMC null mice. Our findings contribute to a growing body of evidence demonstrating that opioid peptides are an integral component of vertebrate retinas, including those of mammals.
PMCID: PMC3095846  PMID: 20533364
retina; β-endorphin; starburst amacrine cell; POMC
15.  Development of Transgenic Papaya through Agrobacterium-Mediated Transformation 
Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.
PMCID: PMC3771462  PMID: 24066284
16.  Overexpression of phyA and appA Genes Improves Soil Organic Phosphorus Utilisation and Seed Phytase Activity in Brassica napus 
PLoS ONE  2013;8(4):e60801.
Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg–1 seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.
PMCID: PMC3616117  PMID: 23573285
17.  Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production 
Plant Methods  2010;6:8.
Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed.
In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2) gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV) 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis.
The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.
PMCID: PMC2845569  PMID: 20222981
18.  The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants 
PLoS ONE  2011;6(4):e19474.
Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs).
Methodology/Principal Findings
Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase.
In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.
PMCID: PMC3084875  PMID: 21559333
19.  Regeneration of Transgenic Rice with Bacterial ipt Gene Driven by Senescence Specific (SAG12) Promoter by Particle Bombardment 
Transgenic rice plants were generated using particle bombardment to introduce the Agrobacterium cytokinin biosynthesis gene driven by Arabidopsis (Arabidopsis thaliana) senescence specific promoter (SAG12) for delaying leaf senescence. Using two plasmids we co-transformed one week old embryogenic calli derived from mature Japonica rice (Oryza sativa) variety Chin Guang. The selectable marker hygromycin phosphotransferase (hph) gene and the reporter gene B-ß-glucuronidase (uidA), both under the control of cauliflower mosaic virus (CaMV) 35S promoter were placed on the same co-integrate vector whereas the cytokinin biosynthesis gene, isopentenyl transferase (ipt) driven by the SAG12 promoter is supplied in another plasmid. A total of 32 transgenic rice plants were regenerated of which 27 plants were randomly selected for histochemical ß-glucuronidase (GUS) assay, PCR and Southern blot analysis. Co-integration frequencies of 88% and 69% were obtained for two linked genes (uidA and hph) and two unlinked genes (hph and ipt gene) respectively in R0 plants. Southern blot analysis confirmed the results of histochemical GUS assay and PCR amplifications. A complex integration pattern for all the transgenes including the multiple copies integration was predominantly observed.
PMCID: PMC3799406  PMID: 24575232
Co-transformation; Particle Bombardment; ipt Gene; SAG12 Promoter; Rice
20.  Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure. 
Nucleic Acids Research  1990;18(21):6319-6324.
Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.
PMCID: PMC332498  PMID: 2243778
21.  Development of a ΔglnA Balanced Lethal Plasmid System for Expression of Heterologous Antigens by Attenuated Vaccine Vector Strains of Vibrio cholerae 
Infection and Immunity  2000;68(1):221-226.
We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a ΔglnA derivative of V. cholerae vaccine strain Peru2. This mutant strain, Peru2ΔglnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1.8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus. Strain Peru2ΔglnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2ΔglnA, Peru2ΔglnA(pKEK71-NotI), Peru2(pTIC5), Peru2ΔglnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2ΔglnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals. However, specific anti-CtxB immune responses in serum (P ≤ 0.05) and bile (P ≤ 0.001) were significantly higher in animals that received Peru2ΔglnA(pTIC5) than in those that received ETR3, confirming the advantage of higher-level antigen expression in vivo. The development of this balanced lethal system thus permits construction and maintenance of vaccine and vector strains of V. cholerae that express high levels of immunogenic antigens from plasmid vectors without the need for antibiotic selection pressure.
PMCID: PMC97124  PMID: 10603391
22.  Astroglial cells in the external granular layer are precursors of cerebellar granule neurons in neonates 
It is well established that cerebellar granule cell precursors (GCPs) initially derive from progenitors in the rhombic lip of the embryonic cerebellar primordium. GCPs proliferate and migrate tangentially across the cerebellum to form the external granule cell layer (EGL) in late embryogenesis and early postnatal development. It is unclear whether GCPs are specified exclusively in the embryonic rhombic lip or whether their precursor persists in the neonate. Using transgenic mice expressing DsRed under the human glial fibrillary acidic protein (hGFAP) promoter, we found 2 populations of DsRed+ cells in the EGL in the first postnatal week defined by bright and faint DsRed fluorescent signal. Bright DsRed+ cells have a protein expression profile and electrophysiological characteristics typical of astrocytes, but faint DsRed+ cells in the EGL and internal granule cell layer (IGL) express markers and physiological properties of immature neurons. To determine if these astroglial cells gave rise to GCPs, we genetically tagged them with EGFP or βgal reporter genes at postnatal day (P)3-P5 using a hGFAP promoter driven inducible Cre recombinase. We found that GFAP promoter+ cells in the EGL are proliferative and express glial and neural stem cell markers. In addition, immature granule cells (GCs) en route to the IGL at P12 as well as GCs in the mature cerebellum, 30 days after recombination, express the reporter protein, suggesting that GFAP promoter+ cells in the EGL generate a subset of granule cells. The identification of glial cells which function as neuronal progenitor cells profoundly impacts our understanding of cellular plasticity in the developing cerebellum.
PMCID: PMC2900521  PMID: 20470892
cerebellum; GFAP; astrocytes; neural stem cells; granule cells; external granule cell layer; Bergmann glial cells; Math1; GABA interneurons
23.  The Short-Chain Fatty Acid Methoxyacetic Acid Disrupts Endogenous Estrogen Receptor-α–Mediated Signaling 
Environmental Health Perspectives  2009;117(11):1702-1706.
Ethylene glycol monomethyl ether (EGME) exposure is associated with impaired reproductive function. The primary metabolite of EGME is methoxyacetic acid (MAA), a short-chain fatty acid that inhibits histone deacetylase activity and alters gene expression.
Because estrogen signaling is necessary for normal reproductive function and modulates gene expression, the estrogen-signaling pathway is a likely target for MAA; however, little is known about the effects of MAA in this regard.
We evaluated the mechanistic effects of MAA on estrogen receptor (ER) expression and estrogen signaling using in vitro and in vivo model systems.
MAA potentiates 17β-estradiol (E2) stimulation of an estrogen-responsive reporter plasmid in HeLa cells transiently transfected with either a human ERα or ERβ expression vector containing a cytomegalovirus (CMV) promoter. This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter. In contrast to its effects on exogenous ER, MAA decreases endogenous ERα expression and attenuates E2-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus.
These results illustrate the importance of careful experimental design and analysis when assessing the potential endocrine-disrupting properties of a compound to ensure biological responses are in concordance with in vitro analyses. Given the established role of the ER in normal reproductive function, the effects of MAA on the endogenous ER reported here are consistent with the reproductive abnormalities observed after EGME exposure and suggest that these toxicities may be due, at least in part, to attenuation of endogenous ER-mediated signaling.
PMCID: PMC2801194  PMID: 20049119
estrogen receptor; estrogen; methoxyacetic acid; short-chain fatty acid
24.  Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices 
Journal of Experimental Botany  2009;60(13):3797-3807.
Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.
PMCID: PMC2736893  PMID: 19574251
Agrobacterium rhizogenes; arbuscular mycorrhiza; composite plants; Glomus intraradices; isoflavanoids; transcript profiling; transmission electron microscopy
25.  Transgene expression and silencing in a tick cell line: a model system for functional tick genomics 
The genome project of the blacklegged tick, Ixodes scapularis provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5,000–10,000 cells and cultures were incubated 2 to 3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis.
PMCID: PMC2581827  PMID: 18722527
Ixodes scapularis; transformation; cell line; RNAi; Sleeping Beauty

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