Rootstocks Overexpressing StNPR1 and StDREB1 Improve Osmotic Stress Tolerance of Wild-Type Scion in Transgrafted Tobacco Plants.

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants; of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.

Immunomodulation in Administration of rAAV: Preclinical and Clinical Adjuvant Pharmacotherapies.

Recombinant adeno-associated virus (rAAV) has attracted a significant research focus for delivering genetic therapies to target cells. This non-enveloped virus has been trialed in many clinical-stage therapeutic strategies but important obstacle in clinical translation is the activation of both innate and adaptive immune response to the protein capsid, vector genome and transgene product. In addition, the normal population has pre-existing neutralizing antibodies against wild-type AAV, and cross-reactivity is observed between different rAAV serotypes. While extent of response can be influenced by dosing, administration route and target organ(s), these pose concerns over reduction or complete loss of efficacy, options for re-administration, and other unwanted immunological sequalae such as local tissue damage. To reduce said immunological risks, patients are excluded if they harbor anti-AAV antibodies or have received gene therapy previously. Studies have incorporated immunomodulating or suppressive regimens to block cellular and humoral immune responses such as systemic corticosteroids pre- and post-administration of Luxturna® and Zolgensma®, the two rAAV products with licensed regulatory approval in Europe and the United States. In this review, we will introduce the current pharmacological strategies to immunosuppress or immunomodulate the host immune response to rAAV gene therapy.

Sharing and Helping: Regularity and Characteristics of Pathogenesis of a Widely Used Transgene Initiated Murine Acute Promyelocytic Leukemia Model.

A transgenic acute promyelocytic leukemia (APL) murine model established by Michael Bishop by cloning a human PML-RARα cDNA into the hMRP8 expression cassette has been widely used in the all-trans retinoid acid and arsenic preparations for the research of APL. However, in the existing literature, the data of regularity and characteristics of the pathogenesis of this model were still missing, which hinder the development of many studies, especially application of new technologies such as single-cell sequencing. Therefore, in this article, we have made up this part of the missing data using an improved APL murine model. We clarified the effects of different inoculation doses on the onset time, latency, morbidity, life span, and proportion of APL cells in peripheral blood (PB), spleen, bone marrow, and so on. The relationship between the proportion of APL cells in the bone marrow, spleen, and PB and organ histological changes was also revealed. These results were a supplement and refinement of this APL model. It would add to the knowledge base of the field and aid in ensuring that accurate models are used for directed interventions. It also provides a great convenience for the researchers who will carry out similar research.

Flavanol-rich lychee fruit extract substantially reduces progressive cognitive and molecular deficits in a triple-transgenic animal model of Alzheimer disease.

Effective treatment to prevent or arrest the advance of Alzheimer disease (AD) has yet to be discovered. We investigated whether OligonolR, an FDA-approved flavanol-rich extract prepared from lychee fruit and green tea, exerted beneficial effects relevant to AD in a triple transgenic male mouse model of AD (3×Tg-AD). At 9 months of age, untreated 3×Tg-AD mice vs. wild-type (WT) controls displayed cognitive deficits in behavioral assays and, at 12 months, elevated levels of hippocampal amyloid beta-protein (Aß), amyloid precursor protein (APP), tau phosphorylation, and pro-inflammatory cytokines. 3×Tg-AD mice given Oligonol showed fewer cognitive deficits and attenuated pathological indices at 12 months. Oligonol treatment of 3×Tg-AD mice modulated expression of some critical brain proteins that involve multiple pathways relevant to mitochondrial dysfunction, proteasomal failure, endoplasmic reticulum (ER) stress and synaptic impairment. Together, these results demonstrate that continuous Oligonol treatment attenuates AD-like pathology and cognitive impairment of 3×Tg-AD mice and set the stage for clinical trials of this flavanol-rich plant extract in patients with early AD.

Establishment of drug screening in human embryonic stem cells based on a high-content screening system.

High-content screening (HCS) systems can be used for high-throughput screening of drugs in human embryonic stem cells (hESCs). However, hESCs require immunofluorescence staining with stemness markers (e.g., Oct-4) prior to HCS, which can be time consuming and labor intensive. In this study, we employed transgenic hESCs with enhanced green fluorescent protein driven by stemness gene Oct-4 promoter (Oct-4-EGFP-H9), in which the colony area and relative green fluorescence area inferred a state of hESC proliferation and stemness, respectively. The Oct-4-EGFP-H9 transgenic hESCs were cultured in mTeSR medium with different concentrations of 5-Fluorouracil (5-FU), vitamin C (VC), or retinoic acid (RA) for 5-7 days, followed by repeated imaging using the HCS system. Finally, the hESC colony area and green fluorescence area were calculated. Results showed that 5-FU treatment markedly reduced colony area in a dose-dependent manner, whereas VC and RA treatments did not. MTT assay and flow cytometry indicated that 5-FU inhibited the proliferation of hESCs significantly, verifying reliability of the data from the HCS system based on colony area analysis. The green fluorescence to total colony area ratio decreased with RA treatment, suggesting that RA significantly promoted differentiation, whereas 5-FU and VC had almost no effect, as verified by quantitative real-time polymerase chain reaction and western blot analysis. In conclusion, our study established a rapid and efficient drug screening system without the requirement of staining based on HCS.

Aggrecanase-selective tissue inhibitor of metalloproteinase-3 (TIMP3) protects articular cartilage in a surgical mouse model of osteoarthritis.

A key feature of osteoarthritis is the gradual loss of articular cartilage and bone deformation, resulting in the impairment of joint function. The primary cause of cartilage destruction is considered to be the presence of elevated proteases, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). However, clinically tested global MMP inhibitors have low efficacy that may be due to their lack of selectivity. We previously demonstrated in vitro that a variant of tissue inhibitor of metalloproteinase-3 ([-1A]TIMP3) inhibits ADAMTSs but not MMPs. In this study, we tested whether the selectivity of [-1A]TIMP3 is beneficial compared with that of the wild-type TIMP3 in preventing or delaying the onset of the degenerative effects in a mouse model of osteoarthritis. We generated transgenic mice that overexpressed TIMP3 or [-1A]TIMP3 driven by a chondrocyte-specific type II collagen promoter. TIMP3 transgenic mice showed compromised bone integrity as opposed to [-1A]TIMP3 mice. After surgically induced joint instability, TIMP3 overexpression proved to be less protective in cartilage destruction than [-1A]TIMP3 at late stages of OA. The selective inhibition of ADAMTSs provides the possibility of modifying TIMP3 to specifically target a class of cartilage-degrading proteinases and to minimize adverse effects on bone and possibly other tissues.

Engineered RNA-binding protein for transgene activation in non-green plastids.

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1-3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.

Multilocation comparison of fruit composition for 'HoneySweet', an RNAi based plum pox virus resistant plum.

'HoneySweet', a transgenic plum (Prunus domestica) resistant to plum pox virus through RNAi, was deregulated in the U.S. in 2011. The compositional study of 'HoneySweet' fruit was expanded to include locations outside of the US as well as utilizing a wide variety of comparators and different collection years to see the variability possible. The results revealed that plums have a wide variation in composition and that variation among locations was greater than variation among cultivars. This was also the case for different years at one location. The results supported the supposition that the transgene and insertion event had no significant effect on the composition of 'HoneySweet' fruit even under virus pressure, and that it fell in the normal range of composition of commercially grown plums. It also suggested that the effect of environment is as great as that of genetics on the fruit composition of plums.

Leaky Expression of the TET-On System Hinders Control of Endogenous miRNA Abundance.

With the ability to affect multiple genes and fundamental pathways simultaneously, miRNA engineering of Chinese Hamster Ovary (CHO) cells has significant advantages over single gene expression or repression. Tight control of these molecular triggers is desirable as it could in theory allow on/off or even tunable regulation of desirable cellular phenotypes. The present study investigated the potential of employing a tetracycline inducible (TET-On) system for conditional knockdown of specific miRNAs but encountered several challenges. The authors show a significant reduction in cell proliferation and culture viability when maintained in media supplemented with the TET-On induction agent Doxycycline at concentrations commonly reported. Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown. This work highlights challenges of the TET-On inducible expression system for controlled manipulation of endogenous miRNAs with two examples; miR-378 and miR-455. The authors suggest a solution involving isolation of highly inducible clones and use a single cell analysis platform to demonstrate the heterogeneity of basal expression and inducibility. Finally, the authors describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.

Transcriptome profiling of transgenic potato plants provides insights into variability caused by plant transformation.

Crop genetic engineering involves transformation in which transgenic plants are regenerated through tissue culture manipulations that can elicit somaclonal variation due to mutations, translocations, and/or epigenetic alterations. Here, we report on alterations in the transcriptome in a panel of transgenic potato plants engineered to be herbicide resistant. Using an inbred diploid potato clone (DMRH S5 28-5), ten single-insert transgenic lines derived from independent Agrobacterium-mediated transformation events were selected for herbicide resistance using an allelic variant of acetolactate synthase (mALS1). Expression abundances of the single-copy mALS1 transgene varied in individual transgenic lines was correlated with the level of phenotypic herbicide resistance, suggesting the importance of transgene expression in transgenic performance. Using RNA-sequencing, differentially expressed genes were identified with the proportion of genes up-regulated significantly higher than down-regulated genes in the panel, suggesting a differential impact of the plant transformation on gene expression activation compared to repression. Not only were transcription factors among the differentially expressed genes but specific transcription factor binding sites were also enriched in promoter regions of differentially expressed genes in transgenic lines, linking transcriptomic variation with specific transcription factor activity. Collectively, these results provide an improved understanding of transcriptomic variability caused by plant transformation.

A combinatorial approach for robust transgene delivery and targeted expression in mammary gland for generating biotherapeutics in milk, bypassing germline gene integration.

Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo ß-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo ß-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo ß-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.

Molecular Approaches for Manipulating Male Sterility and Strategies for Fertility Restoration in Plants.

Usable pollination control systems have proven to be effective system for the development of hybrid crop varieties, which are important for optimal performance over varied environments and years. They also act as a biocontainment to check horizontal transgene flow. In the last two decades, many genetic manipulations involving genes controlling the production of cytotoxic products, conditional male sterility, altering metabolic processes, post-transcriptional gene silencing, RNA editing and chloroplast engineering methods have been used to develop a proper pollination control system. In this review article, we outline the approaches used for generating male sterile plants using an effective pollination control system to highlight the recent progress that occurred in this area. Furthermore, we propose possible future directions for biotechnological improvements that will allow the farmers to buy hybrid seed once for many generations in a cost-effective manner.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

KEY MESSAGE: Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 µM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one- and two-copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.

Evaluation of both targeted and non-targeted cell wall polysaccharides in transgenic potatoes.

In this study, we analyze 31 transgenic lines and their respective untransformed background lines to determine the transgene effects on targeted structures including the pectin components rhamnogalacturonan I (RG-I) and homogalacturonan (HG), neutral side chains (galactan/arabinangalactan), acetylation of pectin, and cellulose level. Modification arising from the pectin backbone- or pectin side chain transgenic lines either increased or decreased the HG:RG-I ratio, side chain length, and methyl esterification of pectin in the tuber cell wall. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level-targeting transgenic lines yielded an unexpectedly high HG:RG-I ratio and longer pectic side chains. These results clearly demonstrate that in effects of a transgene are not restricted to the direct activity of the targeted enzyme but have consequences for the structure of the cell wall matrix. Analysis of whole cell wall structure is therefore necessary to assess the complete effect, direct and indirect, of a transgene.

Event-specific detection of transgenic potato AV43-6-G7 using real-time and digital PCR methods.

BACKGROUND: The isolation of unknown DNA sequences flanked by known sequences is an important task in the event-specific detection of GMOs. None of event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic potato AV43-6-G7. RESULTS: The flanking sequence between the exogenous fragment and recombinant chromosome of this potato was successfully acquired through exogenous gene 5'-RACE. The event-specific primers and Taqman probe were designed to amplify fragments spanning the exogenous DNA and potato genomic DNA. The specific real-time PCR and digital PCR detection methods for AV43-6-G7 potato were established based on primers designed according to the flanking sequences. The detection limit of the qualitative PCR assay was 0.01 % for AV43-6-G7 potato in 100 ng of potato genomic DNA, corresponding to approximately 11.6 copies of the potato haploid genome. The ddPCR assays for Potato AV43-6-G7 achieved a limit of quantification of approximately 58 target copies, with RSD ≤ 25 %. The aLOQ of this system was approximately 1.2 copies. CONCLUSIONS: These results indicated that these event-specific methods would be useful for the identification of potato AV43-6-G7.

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

BACKGROUND/AIMS: Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. METHODS: Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. RESULTS: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. CONCLUSION: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids.

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica.

Mirabilis himalaica is an endangered medicinal plant species in the Tibetan Plateau. The two genes respectively encoding chalcone synthase (MhCHS) and chalcone isomerase (MhCHI) were isolated and characterized from M. himalaica. The sequence analysis revealed that the two genes were similar with their corresponding homologous genes in other plants. The tissue profiles showed that both MhCHS and MhCHI had higher expression levels in roots than in stems and leaves. Transgenic hairy root cultures respectively with overexpressing MhCHS and MhCHI were established. The genomic PCR detection confirmed the authority of transgenic hairy root lines, in which either MhCHS or MhCHI expression levels were much higher than that in non-transgenic hairy root line. Finally, the HPLC detection results demonstrated that the rotenoid contents in MhCHS/MhCHI-transformed hairy root lines were enhanced. This study provided two candidate genes that could be used to genetic engineering rotenoid biosynthesis in M. himalaica and an alternative method to produce rotenoid using transgenic hairy root cultures.

Enhanced production of docosahexaenoic acid in mammalian cells.

Docosahexaenoic acid (DHA), one of the important polyunsaturated fatty acids (PUFA) with pharmaceutical and nutraceutical effects, may be obtained through diet or synthesized in vivo from dietary a-linolenic acid (ALA). However, the accumulation of DHA in human body or other mammals relies on the intake of high dose of DHA for a certain period of time, and the bioconversion of dietary ALA to DHA is very limited. Therefore the mammalian cells are not rich in DHA. Here, we report a new technology for increased production of DHA in mammalian cells. By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO) cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed. The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA) which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase. This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA.