Transgenic Tissues Research Articles

Enhanced drought and salinity tolerance in transgenic mustard [Brassica juncea (L.) Czern & Coss.] overexpressing Arabidopsis group 4 late embryogenesis abundant gene (AtLEA4-1)

Indian mustard [Brassica juncea (L.) Czern & Coss.] is one of the most important oilseed crop worldwide, yet relatively mild drought and salinity stress significantly reduce its growth and yield. Expression of LEA (Late Embryogenesis Abundant) proteins was previously shown to alleviate osmotic stress by stabilizing water status, protecting cytosolic structures and cell through increased membrane and protein stability. We overexpressed Arabidopsis group 4 LEA protein AtLEA4-1 in mustard, resulting the transgenic lines to show enhanced drought and salinity tolerance. Southern hybridization showed the copy number of the gene inserted into the transgenics while, qRT PCR determined the transcript level of the overexpressed gene. Leaf senescence assay showed better adaptability of the transgenics to both salt and PEG stress, as revealed by increased chlorophyll content in the transgenic plants when compared to wild type. The transgenic mustard plants showed better survivability under both drought and salinity stress in soil. Physiological analysis revealed lower levels of lipid peroxidation, hydrogen peroxide and oxygen radical production but higher levels of relative water content (RWC) in T1 transgenic AtLEA4-1 lines with not so significant increase in proline content. Antioxidative enzyme activities showed better adaptation of transgenic plants to drought stress. Even when the biochemical parameters were analysed as a function of RWC, the transgenic plant tissues showed improved adaptation to drought and salinity stress depicting, the functions of LEA proteins as antioxidant and molecular chaperone exerting equivalent impact as that of hydration buffer towards abiotic stress tolerance.

Identification of multiple lipid genes with modifications in expression and sequence associated with the evolution of hydroxy fatty acid accumulation in Physaria fendleri.

Two Brassicaceae species, Physaria fendleri and Camelina sativa, are genetically very closely related to each other and to Arabidopsis thaliana. Physaria fendleri seeds contain over 50% hydroxy fatty acids (HFAs), while Camelina sativa and Arabidopsis do not accumulate HFAs. To better understand how plants evolved new biochemical pathways with the capacity to accumulate high levels of unusual fatty acids, transcript expression and protein sequences of developing seeds of Physaria fendleri, wild-type Camelina sativa, and Camelina sativa expressing a castor bean (Ricinus communis) hydroxylase were analyzed. A number of potential evolutionary adaptations within lipid metabolism that probably enhance HFA production and accumulation in Physaria fendleri, and, in their absence, limit accumulation in transgenic tissues were revealed. These adaptations occurred in at least 20 genes within several lipid pathways from the onset of fatty acid synthesis and its regulation to the assembly of triacylglycerols. Lipid genes of Physaria fendleri appear to have co-evolved through modulation of transcriptional abundances and alterations within protein sequences. Only a handful of genes showed evidence for sequence adaptation through gene duplication. Collectively, these evolutionary changes probably occurred to minimize deleterious effects of high HFA amounts and/or to enhance accumulation for physiological advantage. These results shed light on the evolution of pathways for novel fatty acid production in seeds, help explain some of the current limitations to accumulation of HFAs in transgenic plants, and may provide improved strategies for future engineering of their production.

Gene electrotransfer of plasmids encoding cytokines as an effective immunotherapy approach for melanoma

Abstract Gene electrotransfer (GET) of plasmids encoding cytokines has been shown to generate a potent anti-tumor effect. Effective protocols have been developed delivering the plasmids directly into tumors. To obtain an effective immune response, it is critical to achieve a balance between transgene expression and tissue damage. GET is a reliable and effective physical method for in vivo delivery of plasmid DNA. It is particularly attractive for use in anti-cancer cytokine therapy as it does not incur the side effects associated with conventional protein cytokine therapy. Delivery of plasmids encoding cytokines directly to tumors has been shown by our lab and others to induce not only local immune response, but a systemic one as well. The positive responses were directly related to the ability to achieve the appropriate expression profile following delivery of the plasmid. Interestingly, the electrotransfer parameters were dependent on which cytokine was being delivered. For plasmid encoding IL-12 the correct parameters were 1300 V/cm and 100 us. For IL-15, the appropriate parameters were 600 V/cm and 5 ms. The procedure included injecting the plasmid DNA into established B16.F10 melanoma tumors of C57BL/6 mice and electric pulses applied three times over a one week period. To enhance this immune response, current work is further analyzing the immune response to determine if there is a marker that would indicate appropriate delivery of the plasmid. In addition, combination therapies are being explored to evaluate if the response could be augmented. A more complete understanding of the process and identifying other agents that could further augment this approach would be an important step.

Generation of Soybean (Glycine max) Transient Transgenic Roots

Legumes-because of their nitrogen-fixing capacity-have both ecological and agronomic importance, and are also the major plant protein source for animal consumption. The model legume species are Lotus japonicus, Medicago truncatula, and soybean (Glycine max). These species have sequenced genomes and are amenable to genetic manipulation, as well as to various functional genomic and cell biology approaches. Plant transformation mediated by Agrobacterium is one of the most powerful methods in plant biotechnology. Using the traditional Agrobacterium tumefaciens method, stable transgenic plants take 6 to 12 months to create, depending on species. Besides being time consuming, this approach is often quite laborious. Hence, there is a need for more rapid methods to create transgenic tissues. In the case of roots, this can be done using hairy root transformation mediated by Agrobacterium rhizogenes. This protocol describes a method to generate transgenic soybean roots in as little as 3 weeks. © 2016 by John Wiley & Sons, Inc.

Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy.

A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences.

Targeting insect mitochondrial complex I for plant protection.

SummaryPlant engineered to express double‐stranded RNA (dsRNA) can target the herbivorous insect gene for silencing. Although mounting evidence has emerged to support feasibility of this new pest control technology, field application is slow largely due to lack of potent targets. Here, we show that suppression of the gene encoding NDUFV2, a subunit of mitochondrial complex I that catalyses NADH dehydrogenation in respiratory chain, was highly lethal to insects. Feeding cotton bollworm (Helicoverpa armigera) larvae with transgenic cotton tissues expressing NDUFV2 dsRNA led to mortality up to 80% within 5 days, and almost no larvae survived after 7 days of feeding, due to the altered mitochondrial structure and activity. Transcriptome comparisons showed a drastic repression of dopa decarboxylase genes. Reciprocal assays with Asian corn borer (Ostrinia furnacalis), another lepidopteran species, revealed the sequence‐specific effect of NDUFV2 suppression. Furthermore, the hemipteran lugus Apolygus lucorum was also liable to NDUFV2 repression. These data demonstrate that the mitochondrial complex I is a promising target with both sequence specificity and wide applicability for the development of new‐generation insect‐proof crops.

The heat shock response plays an important role in TDP-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis.

Detergent-resistant, ubiquitinated and hyperphosphorylated Tar DNA binding protein 43 (TDP-43, encoded by TARDBP) neuronal cytoplasmic inclusions are the pathological hallmark in ∼95% of amyotrophic lateral sclerosis and ∼60% of frontotemporal lobar degeneration cases. We sought to explore the role for the heat shock response in the clearance of insoluble TDP-43 in a cellular model of disease and to validate our findings in transgenic mice and human amyotrophic lateral sclerosis tissues. The heat shock response is a stress-responsive protective mechanism regulated by the transcription factor heat shock factor 1 (HSF1), which increases the expression of chaperones that refold damaged misfolded proteins or facilitate their degradation. Here we show that manipulation of the heat shock response by expression of dominant active HSF1 results in a dramatic reduction of insoluble and hyperphosphorylated TDP-43 that enhances cell survival, whereas expression of dominant negative HSF1 leads to enhanced TDP-43 aggregation and hyperphosphorylation. To determine which chaperones were mediating TDP-43 clearance we over-expressed a range of heat shock proteins (HSPs) and identified DNAJB2a (encoded by DNAJB2, and also known as HSJ1a) as a potent anti-aggregation chaperone for TDP-43. DNAJB2a has a J domain, allowing it to interact with HSP70, and ubiquitin interacting motifs, which enable it to engage the degradation of its client proteins. Using functionally deleted DNAJB2a constructs we demonstrated that TDP-43 clearance was J domain-dependent and was not affected by ubiquitin interacting motif deletion or proteasome inhibition. This indicates that TDP-43 is maintained in a soluble state by DNAJB2a, leaving the total levels of TDP-43 unchanged. Additionally, we have demonstrated that the levels of HSF1 and heat shock proteins are significantly reduced in affected neuronal tissues from a TDP-43 transgenic mouse model of amyotrophic lateral sclerosis and patients with sporadic amyotrophic lateral sclerosis. This implies that the HSF1-mediated DNAJB2a/HSP70 heat shock response pathway is compromised in amyotrophic lateral sclerosis. Defective refolding of TDP-43 is predicted to aggravate the TDP-43 proteinopathy. The finding that the pathological accumulation of insoluble TDP-43 can be reduced by the activation of HSF1/HSP pathways presents an exciting opportunity for the development of novel therapeutics.

Imaging of amyloid-β in Alzheimer's disease transgenic mouse brains with ToF-SIMS using immunoliposomes.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer's disease (AD), in transgenic mouse brain tissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid-protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.

Establishment of a Novel Model for Anticancer Drug Resistance in Three-Dimensional Primary Culture of Tumor Microenvironment.

Tumor microenvironment has been implicated in tumor development and progression. As a three-dimensional tumor microenvironment model, air liquid interface (ALI) organoid culture from oncogene transgenic mouse gastrointestinal tissues was recently produced. However, ALI organoid culture system from tissues of colorectal cancer patients has not been established. Here, we developed an ALI organoid model from normal and tumor colorectal tissues of human patients. Both organoids were successfully generated and showed cystic structures containing an epithelial layer and surrounding mesenchymal stromal cells. Structures of tumor organoids closely resembled primary tumor epithelium. Expression of an epithelial cell marker, E-cadherin, a goblet cell marker, MUC2, and a fibroblast marker, vimentin, but not a myofibroblast marker, α-smooth muscle actin (SMA), was observed in normal organoids. Expression of E-cadherin, MUC2, vimentin, and α-SMA was observed in tumor organoids. Expression of a cancer stem cell marker, LGR5 in tumor organoids, was higher than that in primary tumor tissues. Tumor organoids were more resistant to toxicity of 5-fluorouracil and Irinotecan than colorectal cancer cell lines, SW480, SW620, and HCT116. These findings indicate that ALI organoid culture from colorectal cancer patients may become a novel model that is useful for examining resistance to chemotherapy in tumor microenvironment.

Motility: Hirschsprung disease--laying down a suitable path.

For many patients with Hirschsprung disease, the underlying pathogenic mechanisms are unknown. A new study using a novel line of transgenic mice and tissue from patients with Hirschsprung disease suggests that overproduction of collagen VI could contribute to pathogenesis and the increased incidence of Hirschsprung disease in patients with Down syndrome.

Lethality of Mouse Tissue Factor Pathway Inhibitor Gene Disruption Is Rescued By Transgenic Human Tissue Factor Pathway Inhibitor Knock in

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the extrinsic coagulation pathway. It inhibits FXa generation by forming a quaternary complex containing factor VIIa (FVIIa), tissue factor (TF), factor Xa (FXa), and TFPI. Two TFPI isoforms, TFPI alpha (TFPI a) and TFPI beta (TFPI b), have been identified, which differ in their C-terminal part due to alternative mRNA splicing events. TFPI a consists of three Kunitz domains (KD), while TFPI b contains two KDs and a C terminal GPI anchor linking the protein to endothelial cell surface. Deletion of the first Kunitz domain of TFPI, which is present in TFPI a and TFPI b in mice is known to be incompatible with viability due to intrauterine lethality (Huang et al., 1997).Aim: To generate transgenic humanized TFPI mice in which mouse (m)-TFPI is entirely replaced by human (hu)-TFPI, in order to facilitate analysis of specific anti hu-TFPI antagonists without interference from m-TFPI.Methods: Integration of the targeting vector, consisting of the m TFPI signal sequence, followed by the human TFPI cDNA and subsequent breeding analysis, was followed by genomic PCR. A sophisticated breeding strategy was used to entirely delete m-TFPI exon 4, which encodes KD1, in humanized transgenic mice. Expression of hu-/m-TFPI a and b mRNAs was analyzed by reverse transcription, cloning, and sequencing of the obtained DNA fragments. Protein levels of hu- and m-TFPI in plasma of transgenic and wild-type (wt) mice were analyzed using species specific ELISAs. Immunoprecipitation experiments in plasma and various mouse tissues are being performed to obtain more information on the presence and distribution of the hu-TFPI protein in transgenic mice.Results: Homozygous humanized TFPI mice were viable and exhibited no obvious abnormalities. Animals showed normal litter size with equal numbers of female and male pups. Genomic PCRs revealed proper integration of the targeting vector into the mouse chromosome and the homozygous status with the expected deletion of m-TFPI exon 4. Expression analyses of humanized TFPI mice on mRNA level demonstrated the absence of full length m-TFPI a and the presence of the humanized TFPI mRNA. Alternative spliced m-TFPI b messages lacking exons three and four were identified, likely leading to a nonfunctional protein. Full length hu-TFPI a mRNA was detected in various tissues in the humanized TFPI mice. The TFPI protein level in plasma from humanized mice was below the detection limit of the ELISA and at least ~300 fold below that for wt mice.Conclusion: Low levels of hu-TFPI may compensate the function of m-TFPI in vivo and circumvent embryonic lethality. Furthermore, we established a new mouse model which allows the regulation of physiologic and pathologic pathways to be assessed at TFPI plasma concentrations below the limit of detection. DisclosuresHoellriegl:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.

Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells

Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h.

Transgenic studies for modulating terpenoid indole alkaloids pathway in Catharanthus roseus: present status and future options

The anti-neoplastic herb Catharanthus roseus (Madagascar periwinkle; Family Apocynaceae), known to harbor >130 bioactive terpenoid indole alkaloids (TIAs), is in global attention of transgenic research for pathway engineering. This medicinal herb has a long folklore history of its usage in the treatment of several health disorders owing to the presence of a variety of secondary metabolites, particularly the alkaloids, phenolics and anthocyanins. Among its various bioactive molecules, C. roseus is most valued for being the exclusive bio-resource of two of the most effective anti-cancerous drugs—vincristine and vinblastine that are indispensible components of several modern day chemotherapeutic regimens. The in-planta yields of these bioactive TIAs are extremely low (<0.0002 %) owing to very rigid developmental, genetical and environmental regulation controls as a part of plant’s own auto-protection strategies against these cytotoxic molecules. Consequently the high cost of production coupled with increasing market demand for these alkaloidal drugs have made TIAs pathway as one of the preferred target for plant metabolic engineering efforts to boost their commercial production. The transgenic approaches so far applied in C. roseus can be grouped into three major lines of investigations. The first line of efforts have been dominated by the genetic transformation studies carried out with wild type Ti or Ri plasmids to measure the influence of their random insertion on TIAs biogenesis. These studies, besides helping the optimization of basic transformation protocols at different levels of cellular differentiation, have largely contributed towards the better resolution and expression of TIAs pathway architecture at the level of associated genes and enzymes. Second line of metabolic engineering works is being centered around the efforts on preparation of genomic libraries and genome mapping to hunt for TIAs pathway genes and associated transcription regulators/factors, followed by their cloning, insertion and hyper-expression to modulate the carbon flux towards desired end products. Modern day transgenic attempts in C. roseus are largely focused on superimposing the concurrent influences of biotic and abiotic elicitation, T-DNA activation and RNAi or virus induced gene silencing approaches for higher TIAs biogenesis. The pace of transgenic research in C. roseus in last two decade has been rapid and demands periodic compilation and reviewing of published literature to provide a handy status up-date of the subject. The present compilation is an effort in this direction and intends to provide a state-of-art account of various transgenic approaches currently being pursued in this high value herb. More than 130 research papers have appeared on various aspects of transgenic cells, tissues and plant production in C. roseus in 30 years. The bulk of these efforts (64.96 %) were made at the level of Agrobacterium rhizogenes-mediated hairy root cultures, followed by 26.80 % in A. tumefaciens-mediated transformed tissues and 8.24 % in cells/tissue cultures subjected to particle bombardment approach. In the present compilation, these studies are further grouped into specific categories wherein over-expression of TIAs pathway specific genes, transcriptional factor regulation, elicitation/stress superimposition, transgenic plant regeneration, metabolites profiling and gene silencing etc. were investigated in different transformed tissue.

Decoding c-Myc networks of cell cycle and apoptosis regulated genes in a transgenic mousemodel of papillary lung adenocarcinomas.

The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays.Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies.

Transformation of Cichorium intybus with the HvBADH1 gene enhanced the salinity tolerance of the transformants

Drought, salinity, and freezing are three major abiotic stresses that adversely affect plant growth and crop yield. Previous reports have demonstrated that the overexpression of the betaine aldehyde dehydrogenase (BADH) gene can improve the tolerance of plants to osmotic and salinity stresses. In the present study, a novel and atypical BADH gene, HvBADH1, was transferred into Cichorium intybus by an Agrobacterium tumefaciens-mediated method. The integration and expression of HvBADH1 in the transformants were confirmed by PCR, Southern blot, and RT-PCR analysis. The stress tolerance traits of the transgenic plants were investigated by evaluating the phenotype of the transformed and wild-type (WT) plants, by measuring seven physiological indicators associated with stress resistance in plants, such as the K+ and Na+ contents, K+/Na+ ratio, malondialdehyde (MDA) content, chlorophyll content, the content of glycine betaine (GB), and relative conductivity in the leaves of the plants. The results revealed that the transgenic tissues had a better K+ retention ability, a reduced sensitivity to stress-induced hydroxyl radical production, an enhanced protection of the photosynthetic system, 4.92–6.95-fold greater amount of GB, and a higher growth rate compared with the WT plant. Accordingly, we concluded that the overexpression of HvBADH1 not only enhanced the salt tolerance of transgenic Cichorium intybus, but also reduced the cellular membrane damage caused by the salinity stress.

The commonly used eye-specific sev-GAL4 and GMR-GAL4 drivers in Drosophila melanogaster are expressed in tissues other than eyes also.

The binary GAL4-UAS system of conditional gene expression is widely used by Drosophila geneticists to target expression of the desired transgene in tissue of interest. In many studies, a preferred target tissue is the Drosophila eye, for which the sev-GAL4 and GMR-GAL4 drivers are most widely used since they are believed to be expressed exclusively in the developing eye cells. However, several reports have noted lethality following expression of certain transgenes under these GAL4 drivers notwithstanding the fact that eye is not essential for survival of the fly. Therefore, to explore the possibility that these drivers may also be active in tissues other than eye, we examined the expression of UAS-GFP reporter driven by the sev-GAL4 or GMR-GAL4 drivers. We found that both these drivers are indeed expressed in additional tissues, including a common set of specific neuronal cells in larval and pupal ventral and cerebral ganglia. Neither sev nor glass gene has so far been reported to be expressed in these neuronal cells. Expression pattern of sev-GAL4 driver parallels that of the endogenous Sevenless protein. In addition to cells in which sev-GAL4 is expressed, the GMR-GAL4 is expressed in several other larval cell types also. Further, two different GMR-GAL4 lines also show some specific differences in their expression domains outside the eye discs. These findings emphasize the need for a careful confirmation of the expression domains of a GAL4 driver being used in a given study, rather than relying only on the empirically claimed expression domains.

Overexpression of ARBORKNOX1 delays the differentiation of induced somatic sector analysis (ISSA) derived xylem fibre cells in poplar stems

ARBORKNOX1 (ARK1), a poplar functional homolog of the Arabidopsis Class-I KNOTTED- like homeobox (KNOXI) transcription factor SHOOT MERISTEMLESS (STM), has been shown to have a role in maintaining meristematic cells in an undifferentiated state. Studies in which this gene was ectopically overexpressed identified a delay in the differentiation of cambial derivatives. Overexpression of ARK1 in poplar stem sectors, using the induced somatic sector analysis (ISSA) experimental system, was undertaken to further investigate its role in the differentiation of xylem fibre cells. ISSA involves the in vivo transformation of a small number of cambial cells in the stem with a gene of interest giving rise to a discreet transgenic tissue sector that can be directly compared to adjacent non-transgenic fibres to elucidate gene function. Phenotypes obtained were comparable with the proposed role of ARK1, where a delay in the development of the cell wall in differentiating fibre cells of active cambia was observed. Additional evidence suggests a role for ARK1 in the positive regulation of cell viability in the cambium. The significance of dormancy signals in the context of differentiation was also highlighted, as some transgenic fibres failed to fully differentiate prior to the onset of dormancy. Our findings are discussed in the context of wood formation and the use of ISSA as an experimental system.

A Drosophila Model of Epidermolysis Bullosa Simplex

The blistering skin disorder Epidermolysis bullosa simplex (EBS) results from dominant mutations in K5 or K14 genes, encoding the intermediate filament network of basal epidermal keratinocytes. The mechanisms governing keratin network formation and collapse due to EBS mutations remain incompletely understood. Drosophila lacks cytoplasmic intermediate filaments, providing a ‚null’ environment to examine the formation of keratin networks and determine mechanisms by which mutant keratins cause pathology. Here, we report that ubiquitous co-expression of transgenes encoding wild-type human K14 and K5 resulted in the formation of extensive keratin networks in Drosophila epithelial and non-epithelial tissues, causing no overt phenotype. Similar to mammalian cells, treatment of transgenic fly tissues with phosphatase inhibitors caused keratin network collapse, validating Drosophila as a genetic model system to investigate keratin dynamics. Co-expression of K5 and a K14R125C mutant that causes the most severe form of EBS resulted in widespread formation of EBS-like cytoplasmic keratin aggregates in epithelial and non-epithelial fly tissues. Expression of K14R125C/K5 caused semi-lethality; adult survivors developed wing blisters and were flightless due to lack of intercellular adhesion during wing heart development. This Drosophila model of EBS is valuable for the identification of pathways altered by mutant keratins and for development of EBS therapies.

High-frequency plant regeneration from transgenic rice expressing Arabidopsis thaliana Bax Inhibitor (AtBI-1) tissue cultures

Abstract Genetic transformation was affected by material of explant, age of callus, and medium of regeneration. Two rice seed cultivars (Ilpum and Baekjinju) and mediums were investigated in this study for enhancing regeneration of transgenic rice expressed AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor. Regeneration rate of Ilpum rice transformant in gelrite of 5 and 8 g were 27.4% and 18.0%, respectively. In Baekjinju, regeneration rate of transformant was 5.4% and 4.3% in 5 and 8 g gelrite, respectively. The highest number of transformant plant in this study was regenerated from Ilpum cultivar on MS medium (30.4%) and was applied for the subsequent experiment. The callus regeneration rate of transformant were 40.7% in callus infection of up-side, it was higher regeneration then in the down-side (3.9%). The regeneration rate of callus of 25 days and 35 days were 14.7% and 38.6%, respectively. The most important application of this work is in genetic transformation of rice, particularly for improvement transgenic plant tissue culture protocol with high frequency of plant regeneration.Keywords Agrobacterium -mediated, Transformation, Rice, Regeneration, AtBI-1

Secondary siRNAs from Medicago NB-LRRs modulated via miRNA-target interactions and their abundances.

Small RNAs are a class of non-coding RNAs that are of great importance in gene expression regulatory networks. Different families of small RNAs are generated via distinct biogenesis pathways. One such family specific to plants is that of phased, secondary siRNAs (phasiRNAs); these require RDR6, DCL4, and (typically) a microRNA (miRNA) trigger for their biogenesis. Protein-encoding genes are an important source of phasi-RNAs. The model legume Medicago truncatula generates phasiRNAs from many PHAS loci, and we aimed to investigate their biogenesis and mechanism by which miRNAs trigger these molecules. We modulated miRNA abundances in transgenic tissues showing that the abundance of phasiRNAs correlates with the levels of both miRNA triggers and the target, precursor transcripts. We identified sets of phasiRNAs or PHAS loci that predominantly and substantially increase in response to miRNA overexpression. In the process of validating targets from miRNA overexpression tissues, we found that in the miRNA-mRNA target pairing, the 3' terminal nucleotide (the 22nd position), but not the 10th position, is important for phasiRNA production. Mutating the single 3' terminal nucleotide dramatically diminishes phasiRNA production. Ectopic expression of Medicago NB-LRR-targeting miRNAs in Arabidopsis showed that only a few NB-LRRs are capable of phasiRNA production; our data indicate that this might be due to target inaccessibility determined by sequences flanking target sites. Our results suggest that target accessibility is an important component in miRNA-target interactions that could be utilized in target prediction, and the evolution of mRNA sequences flanking miRNA-target sites may be impacted.