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.
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.
Plant defensin; Antimicrobial peptide; Syncytium; Heterodera schachtii; Arabidopsis; Roots; Dpi, days post infection
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.
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.
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.
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.
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.
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.
retina; β-endorphin; starburst amacrine cell; POMC
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.
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.
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.
We synthesized a Yarrowia lipolytica strain overproducing lipase for industrial applications by using long terminal repeat (ζ) of the Y. lipolytica retrotransposon Ylt1 and an allele of URA3 with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a NotI-NotI cassette which contains a defective URA3 allele, a polylinker sequence, and the ζ region for targeting to multiple sites in the genome of the recipient. We inserted the LIP2 gene (encoding extracellular lipase) under the control of the strong POX2 promoter into JMP3 to generate JMP6. The pHSS6 region was removed by NotI digestion prior to transformation. Two Y. lipolytica strains transformed with the JMP6 LIP2 cassette had a mean of 10 integrated copies devoid of the Escherichia coli region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the LIP2 promoter. This work provides a new expression system in Y. lipolytica that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.
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.
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.
cerebellum; GFAP; astrocytes; neural stem cells; granule cells; external granule cell layer; Bergmann glial cells; Math1; GABA interneurons
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.
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.
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.
Aims: To evaluate the protective effectiveness of gloves from occupational exposure to 2-methoxyethanol (2-ME); and to examine the association of 2-methoxyacetic acid (MAA) in urine and plasma collected simultaneously from low 2-ME exposure and high 2-ME exposure workers in a semiconductor copper laminate circuit board manufacturing plant.
Methods: Eight hour time weighted breathing zone monitoring was performed to verify the 2-ME exposure classification between workers in regular and special operations. Urine and plasma samples were simultaneously collected from 74 exposed and 80 non-exposed workers. MAA concentrations in the urine (UMAA) and plasma (PMAA) were measured using previously published methods. Three types of gloves worn by workers (cotton, rubber, and no gloves) were recorded by direct observations in the workplace and validated by person-to-person interview. Protective effectiveness indices (PEI) were used to evaluate the glove effectiveness.
Results: There was no detectable 2-ME/MAA in the air, or in urine and plasma samples in non-exposed workers. The average UMAA and PMAA in special operations were 72.63 mg/g Cr. and 29.72 mg/l, significantly higher than values in regular operations (5.44 mg/g Cr. and 2.58 mg/l, respectively). PMAA showed satisfactory correlation to UMAA in all participants from both regular and special operations. The rubber gloves provided significant reduction in 2-ME uptake, whereas cotton gloves provided little protection with fluctuating effectiveness, based on PEI estimates.
Conclusions: PMAA, similar to UMAA, could serve as a specific biomarker for 2-ME exposure. Wearing impermeable rubber gloves during high risk tasks can reduce major 2-ME exposure. Other improvements, including engineering control, should be provided to diminish worker exposure to 2-ME in occupational environments.
Sleeping Beauty transposon (SB-Tn) has emerged as an important nonviral vector for integrating transgenes into mammalian genomes. We report here a novel dual fluorescent reporter cis SB-Tn system that permitted nonselective fluorescent-activated cell sorting for SB-Tn-transduced K562 erythroid cells. Using an internal ribosome entry site element, the green fluorescent protein (eGFP) was linked to the SB10 transposase gene as an indirect marker for the robust expression of SB10 transposase. Flourescence-activated cell sorting (FACS) by eGFP resulted in significant enrichment (> 60%) of cells exhibiting SB-Tn-mediated genomic insertions and long-term expression of a DsRed transgene. The hybrid erythroid-specific promoter of DsRed transgene was verified in erythroid or megakaryocyte differentiation of K562 cells. Bisulfite-mediated genomic analyses identified different DNA methylation patterns between DsRed+ and DsRed− cell clones, suggesting a critical role in transgene expression. Moreover, although the host genomic copy of the promoter element showed no CpG methylation, the same sequence carried by the transgene was markedly hypermethylated. Additional evidence also suggested a role for histone deacetylation in the regulation of DsRed transgene. The presence of SB transgene affected the expression of neighboring host genes at distances > 45 kb. Our data suggested that fluorescent reporter cis SB-Tn system can be used to enrich mammalian cells harboring SB-mediated transgene insertions. The epigenetic modification detected on the DsRed transgene demonstrated that transgenes inserted by SB could also be selectively modified by host cellular epigenetic systems. In addition, long-range activation of host genes must now be recognized as a potential consequence of an inserted transgene cassette containing enhancer elements.
A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.
Allergy; Expression system; Green biotechnology; Molecular farming; Recombinant protein
Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome.
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.
Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.
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.
To achieve efficient and sustained gene expression, we developed a new lentivirus/adenovirus hybrid vector (LA vector) that encodes sequences required for production of a human immunodeficiency virus-based lentiviral vector (i.e., a lentiviral vector, a gag/pol/rev expression cassette, a tetracycline-inducible envelope cassette, and the tetracycline-inducible transcriptional activator cassette) in a single helper-dependent adenovirus vector backbone. Via either transfection or infection, human cell lines transduced with the LA vector produced a lentiviral vector in a doxycycline-dependent manner at titers up to 105 to 106 green fluorescent protein transducing units per ml, which are comparable to the titers obtained by conventional multiple plasmid transfection methods. Efficient spread and persistent expression of the transgene were observed in cells maintained in long-term culture that had been infected with the LA vector. Furthermore, when cocultured with adherent cells infected with the LA vector, the human T-cell leukemia cell line was successfully transduced with a marker gene. This LA vector possesses the advantages of efficient gene transfer from an adenoviral vector and stable integration from a lentiviral vector; therefore, it might have potential for a variety of gene therapy applications.
Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.
Recombinant human serum albumin (rHSA); Selectively terminable transgenic rice; Purification