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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.
doi:10.1007/s11105-013-0614-z
PMCID: PMC3881577  PMID: 24415845
Transient expression; Recombinant protein; Agroinfiltration; Viral vector; pPZP vector family
2.  Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities 
A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.
doi:10.3390/ijms150915571
PMCID: PMC4200759  PMID: 25192284
Aspergillus nidulans; phy gene; Escherichia coli; polyclonal antibody; transient expression; Agrobacterium infiltration; oral administration
3.  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.
Highlights
► 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.
doi:10.1016/j.plaphy.2011.07.005
PMCID: PMC3185291  PMID: 21813283
Plant defensin; Antimicrobial peptide; Syncytium; Heterodera schachtii; Arabidopsis; Roots; Dpi, days post infection
4.  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.
doi:10.1128/AEM.72.3.2290-2293.2006
PMCID: PMC1393188  PMID: 16517691
5.  Insulin-like Growth Factor 2 is Required for Progression to Advanced Medulloblastoma in patched1 Heterozygous Mice 
Cancer research  2008;68(21):8788-8795.
Medulloblastoma (MB) can arise in the cerebellum due to genetic activation of the Sonic Hedgehog (Shh) signaling pathway. During normal cerebellum development, Shh spurs the proliferation of granule neuron precursors (GNPs), the precursor cells of MB. Mutations in the Shh receptor gene, patched1 (ptc1+/-), lead to increased MB incidence in humans and mice. MB tumorigenesis in mice heterozygous for ptc1+/- shows distinct steps of progression. Most ptc1+/- mice form clusters of pre-neoplastic cells on the surface of the mature cerebellum that actively transcribe Shh target genes. In ∼15% of mice, these pre-neoplastic cells will become fast-growing, lethal tumors. It was previously shown (1) that the loss of function of insulin-like growth factor 2 (igf2) suppresses MB formation in ptc1+/- mice. We found that igf2 is not expressed in preneoplastic lesions but is induced as these lesions progress to more advanced MB tumors. Igf2 is not required for formation of pre-neoplastic lesions, but is necessary for progression to advanced tumors. Exogenous Igf2 protein promoted proliferation of MB precursor cells (GNPs) and a MB cell line, PZp53MED. Blocking igf2 signaling inhibited growth of PZp53MED cells, implicating igf2 as a potential clinical target.
doi:10.1158/0008-5472.CAN-08-2135
PMCID: PMC2597356  PMID: 18974121
hedgehog; igf2; medulloblastoma; patched; math1
6.  Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants 
BMC Biotechnology  2012;12:17.
Background
The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully) appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues.
Results
The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed.
Conclusions
From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.
doi:10.1186/1472-6750-12-17
PMCID: PMC3443454  PMID: 22554231
Endoplasmic reticulum targeting; Fluorescent proteins; GFP; Marker genes; OFP; Orange fluorescent protein; Reporter genes; RFP; Subcellular localization; Transgenic plants; Visual markers
7.  Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in Emerging Model Systems 
PLoS ONE  2011;6(4):e18659.
Background
Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.
Methodology
We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.
Significance
This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles.
doi:10.1371/journal.pone.0018659
PMCID: PMC3077399  PMID: 21533190
8.  High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene 
Biochemistry  2010;49(7):1507.
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.
doi:10.1021/bi9016846
PMCID: PMC2822882  PMID: 20041635
9.  Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses 
Journal of Experimental Botany  2009;60(6):1859-1871.
A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified.
doi:10.1093/jxb/erp072
PMCID: PMC2671631  PMID: 19357433
Arabidopsis thaliana; arginine biosynthesis; citrulline; drought tolerance; N-acetyl-L-glutamate synthase; ornithine; salt stress; seed germination; transgenic plants
10.  The potato R locus codes for dihydroflavonol 4-reductase 
The potato R locus is required for the production of red pelargonidin-based anthocyanin pigments in potato (Solanum tuberosum L.). Red color also requires tissue-specific regulatory genes, such as D (for expression in tuber skin) and F (expression in flowers). A related locus, P, is required for production of blue/purple anthocyanins; P is epistatic to R. We have previously reported that the dihydroflavonol 4-reductase gene (dfr) co-segregates with R. To test directly whether R corresponds to dfr, we placed the allele of dfr associated with red color under the control of the CaMV 35S promoter and introduced it into the potato cultivar Prince Hairy (genotype dddd rrrr P-), which has white tubers and pale blue flowers. Transgenic Prince Hairy tubers remained white, but flower color changed to purple. Three independent transgenic lines, as well as a vector-transformed line, were then crossed with the red-skinned variety Chieftain (genotype D-R-pppp), to establish populations that segregated for D, R, P, and the dfr transgene or empty vector. Markers were used to genotype progeny at D and R. Progeny carrying the empty vector in the genetic background D-rrrr produced white or purple tubers, while progeny with the same genotype and the dfr transgene produced red or purple tubers. HPLC and LC–MS/MS analyses of anthocyanins present in Chieftain and in a red-skinned progeny clone with the dfr transgene in a D-rrrr background revealed no qualitative differences. Thus, dfr can fully complement R, both in terms of tuber color and anthocyanin composition.
doi:10.1007/s00122-009-1100-8
PMCID: PMC2729421  PMID: 19588118
11.  Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome 
PLoS Genetics  2007;3(10):e179.
Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.
Author Summary
The production of transgenic maize has traditionally used techniques that integrate DNA fragments into a host chromosome. This can disrupt important native genes or can lead to poor expression of the added gene; consequently, large numbers of transgenic plants must be screened to find one suitable for commercial use. Further, there is a limit to the amount of DNA that can be integrated, making it difficult to add multiple genes at one time. Here, we describe a new system for delivering genes to maize. We constructed a minichromosome vector that remains separate, or autonomous, from the plant's chromosomes when introduced into maize cells. These minichromosomes were constructed from DNA sequences that naturally occur in maize centromeres, the chromosomal regions needed for inheritance. We characterized the behavior of Maize Minichromosome 1 (MMC1) through four generations, showing that it is efficiently inherited and that the genes it carries are expressed. This work makes it possible to design minichromosomes that carry several genes, enhancing the ability to engineer plant processes, including improving disease resistance, drought tolerance, or the production of complex biochemicals.
doi:10.1371/journal.pgen.0030179
PMCID: PMC2041994  PMID: 17953486
12.  High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns 
Plant Methods  2010;6:18.
Background
Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.
Results
A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes.
Conclusions
Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.
doi:10.1186/1746-4811-6-18
PMCID: PMC2927586  PMID: 20687964
13.  A non-destructive screenable marker, OsFAST, for identifying transgenic rice seeds 
Plant Signaling & Behavior  2011;6(10):1454-1456.
The production of transgenic plants has contributed greatly to plant research. Previously, an improved method for screening transgenic Arabidopsis thaliana seeds using the FAST (Fluorescence-Accumulating-Seed Technology) method and FAST marker was reported. Arabidopsis seeds containing the FAST marker may be visually screened using a fluorescence stereomicroscope or blue LED handy-type instrument. Although the FAST method was originally designed for Arabidopsis screens, this study endeavors to adapt this method for the screening of other plants. Here, an optimized technology, designated the OsFAST method, is presented as a useful tool for screening transgenic rice seeds. The OsFAST method is based on the expression of the OsFAST-G marker under the control of a seed-embryo-specific promoter, similar to the Arabidopsis FAST-G marker. The OsFAST method provides a simple and non-destructive method for identifying transgenic rice seeds. It is proposed that the FAST method is adaptable to various plant species and will enable a deeper analysis of the floral-dip method.
doi:10.4161/psb.6.10.17344
PMCID: PMC3256369  PMID: 21904112
Oryza sativa; oleosin; seed; green fluorescent protein; transformation; screenable marker
14.  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.
doi:10.1016/j.ibmb.2008.07.008
PMCID: PMC2581827  PMID: 18722527
Ixodes scapularis; transformation; cell line; RNAi; Sleeping Beauty
15.  Iron-biofortification in rice by the introduction of three barley genes participated in mugineic acid biosynthesis with soybean ferritin gene 
Iron deficiency is a serious problem around the world, especially in developing countries. The production of iron-biofortified rice will help ameliorate this problem. Previously, expression of the iron storage protein, ferritin, in rice using an endosperm-specific promoter resulted in a two-fold increase in iron concentration in the resultant transgenic seeds. However, further over expression of ferritin did not produce an additional increase in the seed iron concentration, and symptoms of iron deficiency were noted in the leaves of the transgenic plants. In the present study, we aimed to further increase the iron concentration in rice seeds without increasing the sensitivity to iron deficiency by enhancing the uptake and transport of iron via a ferric iron chelator, mugineic acid. To this end, we introduced the soybean ferritin gene (SoyferH2) driven by two endosperm-specific promoters, along with the barley nicotianamine synthase gene (HvNAS1), two nicotianamine aminotransferase genes (HvNAAT-A and -B), and a mugineic acid synthase gene (IDS3) to enhance mugineic acid production in rice plants. A marker-free vector was utilized as a means of increasing public acceptance. Representative lines were selected from 102 transformants based on the iron concentration in polished seeds and ferritin accumulation in the seeds. These lines were grown in both commercially supplied soil (iron-sufficient conditions) and calcareous soil (iron-deficient conditions). Lines expressing both ferritin and mugineic acid biosynthetic genes showed signs of iron-deficiency tolerance in calcareous soil. The iron concentration in polished T3 seeds was increased by 4 and 2.5 times, as compared to that in non-transgenic lines grown in normal and calcareous soil, respectively. These results indicate that the concomitant introduction of the ferritin gene and mugineic acid biosynthetic genes effectively increased the seed iron level without causing iron sensitivity under iron-limited conditions.
doi:10.3389/fpls.2013.00132
PMCID: PMC3653162  PMID: 23675379
anemia; iron; zinc; rice; mugineic acid; biofortification; ferritin; IDS3
16.  Molecular Characterization of Mycoplasma arthritidis Variable Surface Protein MAA2 
Infection and Immunity  1998;66(6):2576-2586.
Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2 genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed in Escherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidis clonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative −10 box. Full-sized recombinant MAA2 was expressed in E. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.
PMCID: PMC108241  PMID: 9596719
17.  Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein 
BMC Biotechnology  2005;5:20.
Background
DsRed the red fluorescent protein (RFP) isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP) for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems.
Results
We have used embryonic stem cell-mediated transgenesis to evaluate mRFP1 in embryonic stem cells and mice. We find that mRFP1 exhibits the most spatially homogenous expression when compared to the native (tetrameric) and variant dimeric forms of DsRed. High levels of mRFP1 expression do not affect cell morphology, developmental potential or viability and fertility of animals. High levels of widespread mRFP1 expression are maintained in a constitutive manner in embryonic stem cells in culture and in transgenic animals. We have used various optical imaging modalities to visualize mRFP1 expressing cells in culture, in embryos and adult mice. Moreover co-visualization of red, green and cyan fluorescent cells within a sample is easily achieved without the need for specialized methodologies, such as spectral deconvolution or linear unmixing.
Conclusion
Fluorescent proteins with excitation and/or emission profiles in the red part of the visible spectrum represent distinct partners, or longer wavelength substitutes for GFP. Not only do DsRed-based RFPs provide a genetically and spectrally distinct addition to the available repertoire of autoflorescent proteins, but by virtue of their spectral properties they permit deeper tissue imaging. Our work in generating CAG::mRFP1 transgenic ES cells and mice demonstrates the developmental neutrality of mRFP1 in an organismal context. It paves the way for the use of DsRed-based monomeric RFPs in transgenic and gene targeted approaches which often necessitate spatially and/or temporally restricted reporter expression. Moreover animals of the CAG::mRFP1 transgenic strain serve as a source of RFP tagged tissue for the derivation of cell lines and explant, transplant and embryo chimera experiments.
doi:10.1186/1472-6750-5-20
PMCID: PMC1192791  PMID: 15996270
18.  Insulated piggyBac vectors for insect transgenesis 
BMC Biotechnology  2006;6:27.
Background
Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken β-globin HS4 insulator function in both Drosophila and mammalian cells.
Results
To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken β-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects.
Conclusion
The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species.
doi:10.1186/1472-6750-6-27
PMCID: PMC1525164  PMID: 16776846
19.  The AAP gene family for amino acid permeases contributes to development of the cyst nematode Heterodera schachtii in roots of Arabidopsis☆ 
Plant Physiology and Biochemistry  2013;70(100):379-386.
The beet cyst nematode Heterodera schachtii is able to infect Arabidopsis plants and induce feeding sites in the root. These syncytia are the only source of nutrients for the nematodes throughout their life and are a nutrient sink for the host plant. We have studied here the role of amino acid transporters for nematode development. Arabidopsis contains a large number of different amino acid transporters in several gene families but those of the AAP family were found to be especially expressed in syncytia. Arabidopsis contains 8 AAP genes and they were all strongly expressed in syncytia with the exception of AAP5 and AAP7, which were slightly downregulated. We used promoter::GUS lines and in situ RT-PCR to confirm the expression of several AAP genes and LHT1, a lysine- and histidine-specific amino acid transporter, in syncytia. The strong expression of AAP genes in syncytia indicated that these transporters are important for the transport of amino acids into syncytia and we used T-DNA mutants for several AAP genes to test for their influence on nematode development. We found that mutants of AAP1, AAP2, and AAP8 significantly reduced the number of female nematodes developing on these plants. Our study showed that amino acid transport into syncytia is important for the development of the nematodes.
Highlights
•Arabidopsis AAP genes for amino acid permeases are strongly expressed in syncytia.•Mutants of AAP1, AAP2, and AAP8 reduced the number of developing female nematodes.•Amino acid transport into syncytia is important for the development of the nematodes.
doi:10.1016/j.plaphy.2013.05.016
PMCID: PMC3737465  PMID: 23831821
Amino acid transporter; Syncytium; Heterodera schachtii; Arabidopsis; Roots
20.  Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression 
PLoS ONE  2012;7(10):e48287.
The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T2 generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.
doi:10.1371/journal.pone.0048287
PMCID: PMC3485231  PMID: 23118971
21.  Novel sulI binary vectors enable an inexpensive foliar selection method in Arabidopsis 
BMC Research Notes  2011;4:44.
Background
Sulfonamide resistance is conferred by the sulI gene found on many Enterobacteriaceae R plasmids and Tn21 type transposons. The sulI gene encodes a sulfonamide insensitive dihydropteroate synthase enzyme required for folate biosynthesis. Transformation of tobacco, potato or Arabidopsis using sulI as a selectable marker generates sulfadiazine-resistant plants. Typically sulI-based selection of transgenic plants is performed on tissue culture media under sterile conditions.
Findings
A set of novel binary vectors containing a sulI selectable marker expression cassette were constructed and used to generate transgenic Arabidopsis. We demonstrate that the sulI selectable marker can be utilized for direct selection of plants grown in soil with a simple foliar spray application procedure. A highly effective and inexpensive high throughput screening strategy to identify transgenic Arabidopsis without use of tissue culture was developed.
Conclusion
Novel sulI-containing Agrobacterium binary vectors designed to over-express a gene of interest or to characterize a test promoter in transgenic plants have been constructed. These new vector tools combined with the various beneficial attributes of sulfonamide selection and the simple foliar screening strategy provide an advantageous alternative for plant biotechnology researchers. The set of binary vectors is freely available upon request.
doi:10.1186/1756-0500-4-44
PMCID: PMC3060128  PMID: 21366919
22.  Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production 
Plant Methods  2010;6:8.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1746-4811-6-8
PMCID: PMC2845569  PMID: 20222981
23.  Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species 
PLoS ONE  2012;7(12):e53023.
We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T0 transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T2 generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.
doi:10.1371/journal.pone.0053023
PMCID: PMC3532468  PMID: 23285250
24.  Cell Wall Ingrowths in Nematode Induced Syncytia Require UGD2 and UGD3 
PLoS ONE  2012;7(7):e41515.
The cyst nematode Heterodera schachtii infects roots of Arabidopsis plants and establishes feeding sites called syncytia, which are the only nutrient source for nematodes. Development of syncytia is accompanied by changes in cell wall structures including the development of cell wall ingrowths. UDP-glucuronic acid is a precursor of several cell wall polysaccharides and can be produced by UDP-glucose dehydrogenase through oxidation of UDP-glucose. Four genes in Arabidopsis encode this enzyme. Promoter::GUS analysis revealed that UGD2 and UGD3 were expressed in syncytia as early as 1 dpi while expression of UGD1 and UGD4 could only be detected starting at 2 dpi. Infection assays showed no differences between Δugd1 and Δugd4 single mutants and wild type plants concerning numbers of males and females and the size of syncytia and cysts. On single mutants of Δugd2 and Δugd3, however, less and smaller females, and smaller syncytia formed compared to wild type plants. The double mutant ΔΔugd23 had a stronger effect than the single mutants. These data indicate that UGD2 and UGD3 but not UGD1 and UGD4 are important for syncytium development. We therefore studied the ultrastructure of syncytia in the ΔΔugd23 double mutant. Syncytia contained an electron translucent cytoplasm with degenerated cellular organelles and numerous small vacuoles instead of the dense cytoplasm as in syncytia developing in wild type roots. Typical cell wall ingrowths were missing in the ΔΔugd23 double mutant. Therefore we conclude that UGD2 and UGD3 are needed for the production of cell wall ingrowths in syncytia and that their lack leads to a reduced host suitability for H. schachtii resulting in smaller syncytia, lower number of developing nematodes, and smaller females.
doi:10.1371/journal.pone.0041515
PMCID: PMC3406070  PMID: 22848518
25.  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.
doi:10.1016/j.mcn.2010.05.001
PMCID: PMC2900521  PMID: 20470892
cerebellum; GFAP; astrocytes; neural stem cells; granule cells; external granule cell layer; Bergmann glial cells; Math1; GABA interneurons

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