Transgenic Tissues Research Articles

Function of the wheat TaSIP gene in enhancing drought and salt tolerance in transgenic Arabidopsis and rice

Microarray analysis of a salt-tolerant wheat mutant identified a gene of unknown function that was induced by exposure to high levels of salt and subsequently denoted TaSIP (Triticum aestivum salt-induced protein). Quantitative PCR analysis revealed that TaSIP expression was induced not only by salt, but also by drought, abscisic acid (ABA), and other environmental stress factors. Transgenic rice plants that expressed an RNA interference construct specific for a rice gene homologous to TaSIP was more susceptible to salt stress than wild-type rice plants. Subcellular localization studies showed that the TaSIP localized to the cell membrane. Under conditions of salt and drought stress, transgenic Arabidopsis plants that overexpressed TaSIP showed superior physiological properties compared with control plants, including lower Na(+) content and upregulation of several stress resistance genes. Staining of transgenic tissues with β-glucuronidase (GUS) failed to indicate tissue-specific activity of the full-length TaSIP promoter. Quantitative analysis of GUS fluorescence in transgenic plants treated with ABA or salt stress revealed that the region 1,176-1,410 bp from the start codon contained an ABA-responsive element and that the region 579-1,176 bp from the start codon upstream of the exon contained a salt-stress-responsive element. Based on these results, we conclude that the key part of the TaSIP gene is the region of its promoter involved in salt tolerance.

The pJan25 vector series: An enhancement of the Gateway-compatible vector pGWB533 for broader promoter testing applications

Agrobacterium-mediated transformation of plants has enhanced our ability to progress more rapidly in plant genetic engineering. Development of binary vectors for Agrobacterium has played a major role in advancing plant biology. Here, we report new features added to the Gateway-compatible vector pGWB533 for promoter testing with the reporter gene encoding β-glucuronidase (GUS). The original vector contains the spectinomycin/streptomycin adenylyltransferase (aadA) gene for bacterial selection and the hygromycin phosphotransferase gene (hpt) for transformed plant selection. However, some bacterial strains used to transform plants, such as Agrobacterium rhizogenes strain K599, have elevated tolerance to spectinomycin and streptomycin, thus making bacterial selection of pGWB533 inefficient. Although pGWB533 confers chemical selection for transgenic plants using hygromycin resistance, the plasmid has no visual marker that enables visual selection of transformed plants or transgenic tissue. In this regard, adding a gene to constitutively express green fluorescent protein (eGFP) makes it easier to visually select the transformed tissue and trim out the non-transformed.In this report we describe a series of vectors, pJan25S (NCBI: KC416200), pJan25T (NCBI: KC416201) and pJan25X (NCBI: KC416202), that are enhancements of pGWB533 for promoter testing. All three vectors contain the gene encoding eGFP as a visual marker for transformed tissue. However, in pJan25S and pJan25T, eGFP is controlled by the rolD promoter for root-specific expression, while in pJan25X it is controlled by the CaMV35S promoter for constitutive expression in all plant tissues. Spectinomycin and streptomycin resistance remains in pJan25S for bacterial selection; however, pJan25T and pJan25X contain the gene encoding tetracycline resistance (tet) for bacterial selection. These changes resulted in enhanced vectors with better visual and chemical selection that should have broad application in promoter studies.

Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms

Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, expression and developmental regulation of myofilament proteins of mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues using a lacZ reporter gene driven by a cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images, and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in precise synchrony with that in the heart. However, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically pre-set developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart.

Decreased saturated fatty acids, total cholesterol and LDL-C in sdd17 mice

SDD17, a delta-15 desaturase from the fungus Saprolegnia can convert arachidonic acid to eicosapentanoic acid in yeast, plant embryos, and mammalian cells. Here, we generated transgenic mice that carried two copies of codon-optimized sdd17 cDNA within a non-coding domain of chromosome 6. RT-PCR analysis revealed that the foreign gene was expressed in the transgenic tissues. Gas chromatography showed that the levels of total unsaturated fatty acids in muscle, liver, and spleen tissues were significantly (p<0.05) increased in transgenic mice compared to non-transgenic mice at 3 or 8 weeks of age. In addition, the serum concentrations of total cholesterol and low-density lipoprotein cholesterol in transgenic females, but not in males, were significantly lower than those in sex-matched non-transgenic mice. These results suggest that endogenous sdd17 expression is beneficial for mammalian health and that its effects on fatty acid profiles may differ between sexes.

Thirty years of transgenic research in plants

In 1983, the first transgenic tissues and plants were generated by means of disarmed Agrobacterium tumefaciens strains, in which the oncogenes had been replaced by antibiotic resistance markers. Hence, this Special Issue of The International Journal of Developmental Biology celebrates 30 years of transgenic research in plants! Eminent scientists working in the field of plant transformation or plant biotechnology have contributed to this publication and reviewed the state of the art of their particular subdomain or summarized the importance of transgenic research in the discovery of new mechanisms and the establishment of an entirely new field, such as epigenetics.

Neurites containing the neurofilament-triplet proteins are selectively vulnerable to cytoskeletal pathology in Alzheimer’s disease and transgenic mouse models

Amyloid-β plaque accumulation in Alzheimer’s disease (AD) is associated with dystrophic neurite (DN) formation and synapse loss in principal neurons, but interneuron pathology is less clearly characterized. We compared the responses of neuronal processes immunoreactive for either neurofilament triplet (NF+) or calretinin (CR+) to fibrillar amyloid (Aβ) plaques in human end-stage and preclinical AD, as well as in APP/PS1 and Tg2576 transgenic mouse AD models. Neurites traversing the Aβ plaque core, edge, or periphery, defined as 50, 100, and 150% of the plaque diameter, respectively, in human AD and transgenic mouse tissue were compared to age-matched human and wild-type mouse controls. The proportion of NF+ neurites exhibiting dystrophic morphology (DN) was significantly larger than the proportion of dystrophic CR+ neurites in both human AD and transgenic mice (p < 0.01). Additionally, the number of NF+, but not CR+, DNs, correlated with Aβ plaque size. We conclude that CR+ interneurons appear to be more resistant than NF+ neurons to AD-mediated cytoskeletal pathology.

Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

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.

The Complex PrPc-Fyn Couples Human Oligomeric Aβ with Pathological Tau Changes in Alzheimer's Disease

Amid controversy, the cellular form of the prion protein PrP(c) has been proposed to mediate oligomeric amyloid-β (Aβ)-induced deficits. In contrast, there is consistent evidence that the Src kinase Fyn is activated by Aβ oligomers and leads to synaptic and cognitive impairment in transgenic animals. However, the molecular mechanism by which soluble Aβ activates Fyn remains unknown. Combining the use of human and transgenic mouse brain tissue as well as primary cortical neurons, we demonstrate that soluble Aβ binds to PrP(c) at neuronal dendritic spines in vivo and in vitro where it forms a complex with Fyn, resulting in the activation of the kinase. Using the antibody 6D11 to prevent oligomeric Aβ from binding to PrP(c), we abolished Fyn activation and Fyn-dependent tau hyperphosphorylation induced by endogenous oligomeric Aβ in vitro. Finally, we showed that gene dosage of Prnp regulates Aβ-induced Fyn/tau alterations. Together, our findings identify a complete signaling cascade linking one specific endogenous Aβ oligomer, Fyn alteration, and tau hyperphosphorylation in cellular and animal models modeling aspects of the molecular pathogenesis of Alzheimer's disease.

Extracorporeal Xenoperfusion of hCD46/HLA-E Double Transgenic Pig Limbs: Early Immunologic Effects in Composite Tissue Xenografts

Background: An ex vivo model of porcine limb perfusion with whole, anticoagulated human blood was developed to study the early immunologic effects of human blood on hCD46/HLA-E double transgenic porcine tissue avoiding hyperacute rejection. Methods: Six forelimbs (2 wild type & 4 transgenic pigs) were amputated, 3 plexus nerves preserved, and one artery and two veins were cannulated and connected extracorporeally to a specially designed extracorporeal perfusion circuit. The semi-open perfusion circuit was established using a pediatric rotation pump, an oxygenator, a reservoir and a collection bag. Xenogeneic extremity perfusion was performed with whole, heparinanticoagulated human blood. The perfusion parameters (arterial blood gas, response to nerve stimulation etc.) were monitored throughout the experiment. Results: The extracorporeal perfusion with whole human blood in both wild type and transgenic pig extremities is feasible and could be maintained for up to 18h. Xenograft motor activation by direct nerve stimulation was possible throughout the whole extracorporeal perfusion period in both composite tissue xenograft types. No hyperacute rejection was observed macroscopically and by histology. Arterial blood gas analysis demonstrated a stable perfusion, but high lactate levels were recorded. Conclusion: The presented extracorporeal perfusion model is applicable to wild-type and transgenic pigs and offers the possibility to study early immunologic reactions of composite tissue xenografts which include donor type bone marrow.

Characterizing the Mechanism of Action of Double-Stranded RNA Activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

Culturing of transgenic mice liver tissue slices in three-dimensional microfluidic structures of PEG-DA (poly(ethylene glycol) diacrylate)

Abstract The bioreactors with an array of multiple wells favoring the maintenance of the three-dimensional (3-D) liver tissue cultures under continuous perfusion have been developed. All bioreactors were fluidically connected to each other. Each bioreactor in the array contains the poly(ethylene glycol) diacrylate (PEG-DA) microstructures, cultured with the mesothelial cells that support the formation of 3-D environment. The mesothelial cells surrounding liver tissue whose primary functions in vivo are to provide a protective adhesive surface and help in tissue repair. The tissue units were continuously perfused with cell culture medium in the bioreactor. After twelve days of culture, the liver tissue surrounded by the mesothelial cells seeded in the perfused multiwell reactor remained functionally viable as assessed by H&E (hematoxylin and eosin) stain and TUNEL (Terminal deoxynucleotidyl transferase (dUTP) nick end labeling) assay examination. The liver tissue shows intact architecture and enhanced viability compared with those in conventional culture dish and incubation systems. The hepatitis B surface antigen (HBsAg) expression of the liver tissue cultured in our bioreactor was also much better when compared to the conventional static culture method. The use of primary liver sample provides more relevant experimental system and potentially replaces the animal based models.

Localization of cholesterol, amyloid and glia in Alzheimer’s disease transgenic mouse brain tissue using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and immunofluorescence imaging

The spatial distributions of lipids, amyloid-beta deposits, markers of neurons and glial cells were imaged, at submicrometer lateral resolution, in brain structures of a mouse model of Alzheimer's disease using a new methodology that combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) and confocal fluorescence microscopy. The technology, which enabled us to simultaneously image the lipid and glial cell distributions in Tg2576 mouse brain structures, revealed micrometer-sized cholesterol accumulations in hippocampal regions undergoing amyloid-beta deposition. Such cholesterol granules were either associated with individual amyloid deposits or spread over entire regions undergoing amyloidogenesis. Subsequent immunohistochemical analysis of the same brain regions showed increased microglial and astrocytic immunoreactivity associated with the amyloid deposits, as expected from previous studies, but did not reveal any particular astrocytic or microglial feature correlated with cholesterol granulation. However, dystrophic neurites as well as presynaptic vesicles presented a distribution similar to that of cholesterol granules in regions undergoing amyloid-beta accumulation, thus indicating that these neuronal endpoints may retain cholesterol in areas with lesions. In conclusion, the present study provides evidence for an altered cholesterol distribution near amyloid deposits that would have been missed by several other lipid analysis methods, and opens for the possibility to study in detail the putative liaison between lipid environment and protein structure and function in Alzheimer's disease.

A Cross-Platform Comparison of Genome-Wide Expression Changes of Laser Microdissected Lung Tissue of C-Raf Transgenic Mice Using 3′IVT and Exon Array

Microarrays are widely used to study genome-wide gene expression changes in different conditions most notably disease, growth, or to investigate the effects of drugs on entire genomes. While the number and gene probe sequences to investigate individual gene expression changes differs amongst manufactures, the design for all of the probes is biased towards the 3′ region. With the advent of exon arrays, transcripts of any known or predicted exon can be investigated to facilitate the study of genome-wide alternative splicing events. Thus, the use of exon arrays provides unprecedented opportunities in gene expression studies. However, it remains a major challenge to directly compare gene expression data derived from oligonucleotide to exon arrays. In the present study, genome-wide expression profiling of Laser Micro-dissected Pressure Catapulted (LMPC) samples of c-Raf mouse lung adenocarcinoma, dysplasia, unaltered transgenic and non-transgenic tissues was performed using the Affymetrix GeneChip Mouse Genome 430 2.0 Array and whole genome Mouse Exon 1.0 ST Array. Based on individual group comparisons 52 to 83% of regulated genes were similar in direction, but fold changes of regulated genes disagreed when data amongst the two platforms were compared. Furthermore, for 27 regulated genes opposite direction of gene expression was observed when the two platforms were compared pointing to the need to assess alternative splicing events at the 3′ end. Taken collectively, exon arrays can be performed even with laser microdissected samples but fold change gene expression changes differ considerably between 3′IVT array and exon arrays with alternative splicing events contributing to apparent differences in gene expression changes.

α-1 Antitrypsin Promotes Semimature, IL-10–Producing and Readily Migrating Tolerogenic Dendritic Cells

Tolerogenic IL-10-positive CCR7-positive dendritic cells (DC) promote T regulatory (Treg) cell differentiation upon CCR7-dependent migration to draining lymph nodes (DLN). Indeed, in human DC deficiencies, Treg levels are low. α-1 antitrypsin (AAT) has been shown to reduce inflammatory markers, promote a semimature LPS-induced DC phenotype, facilitate Treg expansion, and protect pancreatic islets from alloimmune and autoimmune responses in mice. However, the mechanism behind these activities of AAT is poorly understood. In this study, we examine interactions among DC, CD4(+) T cells, and AAT in vitro and in vivo. IL-1β/IFN-γ-mediated DC maturation and effect on Treg development were examined using OT-II cells and human AAT (0.5 mg/ml). CCL19/21-dependent migration of isolated DC and resident islet DC was assessed, and CCR7 surface levels were examined. Migration toward DLN was evaluated by FITC skin painting, transgenic GFP skin tissue grafting, and footpad DC injection. AAT-treated stimulated DC displayed reduced MHC class II, CD40, CD86, and IL-6, but produced more IL-10 and maintained inducible CCR7. Upon exposure of CD4(+) T cells to OVA-loaded AAT-treated DC, 2.7-fold more Foxp3(+) Treg cells were obtained. AAT-treated cells displayed enhanced chemokine-dependent migration and low surface CD40. Under AAT treatment (60 mg/kg), DLN contained twice more fluorescence after FITC skin painting and twice more donor DC after footpad injection, whereas migrating DC expressed less CD40, MHC class II, and CD86. Intracellular DC IL-10 was 2-fold higher in the AAT group. Taken together, these results suggest that inducible functional CCR7 is maintained during AAT-mediated anti-inflammatory conditions. Further studies are required to elucidate the mechanism behind the favorable tolerogenic activities of AAT.

Development of the “Three-step MACS”: a Novel Strategy for Isolating Rare Cell Populations in the Absence of Known Cell Surface Markers from Complex Animal Tissue

To circumvent the difficulty of isolating specific cell populations by MACS from dissociated complex animal tissue, when their proportions reached levels similar to that of the background, we developed the "Three-step MACS" strategy. Cells of interest are defined by their expression of a particular gene(s) of interest rather by than their natural cell surface markers or size. A two-component transgenic cell surface protein, for two sequential rounds of MACS, is expressed under the promoter control of the endogenous gene of interest by means of gene targeting and the generation of transgenic tissue. An initial step to remove dead cells is also used. Here, we describe proof-of-concept experiments, using the biotin acceptor peptide (BAP)-low-affinity nerve growth factor receptor as the two-component protein. The first component, the BAP, can be biotinylated in specific subsets of cells expressing a particular gene by expressing the biotinylating enzyme, hBirA = humanized BirA (hBirA), under the promoter control of another gene defining the specific subpopulation. We showed that a rare population of cells (1.1% of the 13.5 days postcoital mouse embryo) could be enriched to a sufficiently high purity (84.4%). From another sample with 0.1% of our cells of interest, we achieved a 40.3% pure sample. The low cost, speed, and technical ease of the Three-step MACS also make it scalable and hence, an ideal method for preparing sufficient quantities of biological samples for sensitive, high-throughput assays.

A molecular framework for risk assessment of a virus-tolerant transgenic pepper line

The Cucumber mosaic virus coat protein (CMV-CP) gene-transgenic pepper lines exhibit high tolerance to Cucumber mosaic virus (CMV) strains. In this study, E7, one of the CMVP0-CP transgenic chili pepper events selected by screening was further characterized. Southern blotting and inverse PCR analysis revealed that the E7 event contains a single copy of the inserted gene cassette whose flanking sequences appear to be noncoding and intergenic. We searched for pepper-specific DNA sequence candidates as an endogenous reference gene for GM-pepper detection. We found that only one copy of CaSIG4 and lipocalin genes are present in the pepper genome and their sequences were determined to be pepper-specific. The characterization of the genomic sequences flanking the transgene, as well as the availability of the pepper-specific single copy CaSIG4 and lipocalin genes as endogenous reference genes, enabled the design of E7-event-specific PCR-based quantitative detection methods. The CMV-CP protein levels in the CMV-inoculated wild-type pepper tissues were approximately 60 times higher than those in the uninoculated and CMV-inoculated E7 pepper tissues. These results suggested that the amount of CMV-CP expressed in transgenic pepper tissue was negligible relative to the amount of CMV-CP in the virus-infected wild-type pepper consumed by human beings. This work may prove useful for risk assessment studies of transgenic pepper lines. Furthermore, the characterized single copy genes, lipocalin and CaSIG4, may be used to develop a method to detect gene copy number variations in the pepper genome.

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall

Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants. Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests. An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3-4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass. This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel production.

Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

BackgroundThe 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.ResultsThe 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.ConclusionsFrom 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.

Fruit‐specific overexpression of wound‐induced tap1 under E8 promoter in tomato confers resistance to fungal pathogens at ripening stage

Based on high economic importance and nutritious value of tomato fruits and as previous studies employed E8 promoter in fruit ripening-specific gene expression, we have developed transgenic tomato plants overexpressing tomato anionic peroxidase cDNA (tap1) under E8 promoter. Stable transgene integration was confirmed by polymerase chain reaction (PCR) and Southern analysis for nptII. Northern blotting confirmed elevated tap1 levels in the breaker- and red-ripe stages of T(1) transgenic fruits, whereas wild-type (WT) plants did not show tap1 expression in these developmental stages. Further, tap1 expression levels were significantly enhanced in response to wounding in breaker- and red-ripe stages of transgenic fruits, whereas wound-induced expression of tap1 was not detected in WT fruits. Confocal microscopy revealed high accumulation of phenolic compounds at the wound site in transgenic fruits suggesting a role of tap1 in wound-induced phenolic polymerization. Total peroxidase activity has increased remarkably in transgenic pericarp tissues in response to wounding, while very less or minimal levels were recorded in WT pericarp tissues. Transgenic fruits also displayed reduced post-harvest decay and increased resistance toward Alternaria alternata and Fusarium solani infection with noticeable inhibition in lesion formation. Conidiospore germination and mycelial growth of F. solani were severely inhibited when treated with E8-tap1 fruit extracts compared to WT fruits. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed reduced spore viability when incubated in E8-tap1 fruit extracts. Thus, fruit-specific expression of tap1 using E8 promoter is associated with enhanced total peroxidase activity and high phenolic accumulation in fruits with minimized post-harvest deterioration caused by wounding and fungal attack in tomato fruits.

Abstract 1950: LHRH Peptide decorated cross-linked micelles for the targeted drug delivery to breast cancer

Abstract We have recently developed a series of micellar nanoparticles (NPs) based on linear polyethylene glycol (PEG) and dendritic cholic acids (CA) block copolymer (telodendrimer), for the targeted delivery of chemotherapeutic drugs in the treatment of cancers. The tumor targeting specificity and efficiency of drug-loaded NPs might be further improved by the decoration of cancer cell surface targeting ligands. LHRH (Luteinizing hormone releasing hormone), a natural hormone peptide, is able to bind specifically to the LHRH receptor, which has been reported to be over-expressed in more than 50% breast cancer patients. In the present study, we incorporated the passive targeting of our micellar NPs and the active targeting of LHRH peptide into a single platform with an attempt to further improve the therapeutic index of NPs delivered drugs in the treatment of breast cancer. We synthesized the LHRH peptide on the tentagel resin bead via the solid-phase peptide synthesis method, and demonstrated that human breast cancer cell lines MDA-MB-231 and MDA-MB-435 were able to bind strongly with LHRH peptide beads. Besides breast cancer cell lines, immunohistochemistry staining further demonstrated LHRH peptide was able to specifically bind to human breast tumor tissue as well as transgenic murine mammary carcinoma tissue, but not to normal breast tissue. Alkyne-containing LHRH peptides were successfully conjugated to the azide group at the distal PEG terminus of PEG5k-Cys4-L8-CA8 telodendrimer (cross-linked micelle forming unit) via the “click chemistry”. LHRH peptide ligand significantly enhanced the intracellular delivery of cross-linked micelles in both MDA-MB-231 and MDA-MB-435 breast cancer cells. In addition, the enhanced uptake of LHRH-targeted micelles in those cells can be inhibited by the addition of access of LHRH antibody, indicating the LHRH receptor-medicated internalization. The co-localization of internalized FITC-labeled LHRH-micelles with lysosomal compartment in human breast cancer cells were also observed, suggesting that the endocytosis pathway is likely to be involved in the internalization process. Furthermore, when loaded with paclitaxel (PTX), LHRH-micelles had significantly higher in vitro cellular toxicities against breast cancer cells as compared with the non-targeted micelles. The in vivo biodistribution, tumor targeting specificity and therapeutic efficacies of LHRH targeted micelles in MDA-MB-231 xenograft mouse model are currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1950. doi:1538-7445.AM2012-1950