DsRed Expression Research Articles

A Fast, Efficient, and Tissue-Culture-Independent Genetic Transformation Method for Panax notoginseng and Lilium regale.

The Agrobacterium-based transgenic technique is commonly used for gene function validation and molecular breeding. However, it is not suitable for plants with a low regeneration capacity or a low transformation rate, such as Panax notoginseng (Burk) F.H. Chen and Lilium regale Wilson. In this study, a novel Agrobacterium transformation method based on injection in the meristems was developed using P. notoginseng and L. regale as experimental models. PCR analysis confirmed the successful integration of the reporter gene DsRed2 (Discosoma striata red fluorescence protein 2) into the genome of two experimental models. QRT-PCR and Western blot analysis demonstrated the transcriptional and translational expression of DsRed2. Additionally, laser confocal microscopy confirmed the significant accumulation of the red fluorescent protein in the leaves, stems, and roots of transformed P. notoginseng and L. regale. Most importantly, in the second year after injection, the specific bright orange fluorescence from DsRed2 expression was observed in the transgenic P. notoginseng and L. regale plants. This study establishes a fast, efficient, and tissue-culture-independent transgenic technique suitable for plants with a low regeneration capacity or a low transformation rate. This technique may improve the functional genomics of important medicinal and ornamental plants such as P. notoginseng and L. regale, as well as their molecular breeding.

Abstract 2880: Radiation induces myofibroblast differentiation of pericytes

Abstract Radiation not only kills cancer cells directly through DNA damage, but also indirectly by inducing vascular changes. Endothelial cells (ECs) in small blood vessels and capillaries connect with pericytes, a type of mural cells which are important for vascular stability and structure. Effects of radiation on tumor vasculature are critical for the therapeutic efficacy, however, its impacts on pericytes remain largely unknown. In this study, we focused on the effect of radiation on pericytes and sought to elucidate the mechanism by which radiation affects pericytes. We show 20Gy for C3H10T1/2 (10T1/2) mouse embryonic mesenchymal pericyte precursor cells and 10Gy for human pericytes from placenta (hPC-PL) cells induces pericyte differentiation and activation of adhesion molecules. Radiation enhanced lymphocyte adhesion and decreased permeability in co-culture of ECs and pericytes primarily through endothelial cells, not via pericytes. Furthermore, radiation promoted transdifferentiation of pericytes into myofibroblasts. Supernatant from irradiated cells in combination with a TGFβ inhibitor showed that TGFβ signaling is involved in pericyte differentiation by radiation but not all pericyte marker genes are regulated by TGFβ. Therefore, we performed phospho-kinase array analysis and revealed Akt signaling pathway activation 10 minutes following radiation, which was verified by the phosphorylation of GSK3β and PRAS40. Along with this, generation of intracellular reactive oxygen species (ROS) occurs as early as 5 min following radiation. Inhibitors of Akt and ROS suppress the expression of myofibroblast markers increased by radiation. To investigate whether radiation induces pericyte differentiation into myofibroblast in tumors, we employed NG2DsRedBAC (NG2DsRed) transgenic mice where DsRed is expressed under the control of NG2 promoter to trace pericytes. The endogenous expression of DsRed was detected in pericytes in the tumors implanted to NG2DsRed mice. Flow cytometry analysis of tumors harvested 7 days after radiation shows radiation increased vimentin+ cells and decreased DsRed+ cells, which indicates radiation induced pericyte differentiation into myofibroblast. In conclusion, radiation promotes pericyte maturation and transdifferentiation into myofibroblast via Akt signaling. Further study using Akt inhibitor with radiation to tumor-bearing N2DsRed mice will be warranted. Citation Format: Tomoko Yamazaki, Kelley R. Jordan, Marka R. Crittenden, Michael J. Gough, Kristina H. Young. Radiation induces myofibroblast differentiation of pericytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2880.

Analysis of Notch1 signaling in mammalian sperm development

ObjectiveA mammalian Delta-Notch signaling component, Notch1, has been suggested for its expression during the normal sperm development although its conditional deletion caused no apparent abnormalities. Since we established our original transgenic mouse system that enabled labeling of past and ongoing Notch1 signaling at a cellular level, we tried to validate that observation in vivo. Our transgenic mouse system used Cre/loxP system to induce tandem dsRed expression upon Notch1 signaling.ResultsTo our surprise, we were unable to observe tandem dsRed expression in the seminiferous tubules where the sperms developed. In addition, tandem dsRed expression was lacking in the somatic cells of the next generation in our transgenic mouse system, suggesting that sperms received no Notch1 signaling during their development. To validate this result, we conducted re-analysis of four single-cell RNA-seq datasets from mouse and human testes and showed that Notch1 expression was little in the sperm cell lineage. Collectively, our results posed a question into the involvement of Notch1 in the normal sperm development although this observation may help the interpretation of the previous result that Notch1 conditional deletion caused no apparent abnormalities in murine spermatogenesis.

Sex separation by body color via a W-chromosome-linked transgene

Sex separation processes are important for commercial insect production and sterile insect techniques. Here, we describe the transgenic insertion of a DsRed expression cassette driven by the enhancer HR3 and strong promoter IE1 into the silkworm W chromosome as a dominant visible marker of sex separation. The obtained transgenic lines showed female-specific body color visible to the naked eye at the second- to fifth-instar larval, pupal and adult stages, and their performance traits were comparable to those of a nontransgenic practical silkworm variety. This strategy can greatly facilitate the sex separation of silkworms for male-only rearing and to obtain hybrids while avoiding sibling mating, and it can also be applied to the sex separation of other light-colored insects.

Intraplacental injection of AAV9-CMV-iCre results in the widespread transduction of multiple organs in double-reporter mouse embryos.

Adeno-associated virus serotype 9 (AAV9) has become a popular tool for gene transfer because of its ability to cross the blood-brain barrier and efficiently transduce genetic material into a variety of cell types. The study utilized GRR (Green-to-Red Reporter) mouse embryos, in which the expression of iCre results in the disappearance of Green Fluorescent Protein (GFP) expression and the detection of Discosoma sp. Red Fluorescent Protein (DsRed) expression by intraplacental injection. Our results demonstrate that AAV9-CMV-iCre can transduce multiple organs in embryos at developmental stages E9.5-E11.5, including the liver, heart, brain, thymus, and intestine. These findings suggest that intraplacental injection of AAV9-CMV-iCre is a viable method for the widespread transduction of GRR mouse embryos.

Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines.

Simple SummaryBombyx mori is a lepidopteran insect with economic value. Its genetic background is clear, and genome sequence is relatively complete, but the function of many genes has not been determined. The genetic code expansion system has become an important means of gene function research. In this study, a genetic code expansion system suitable for B. mori cells was established. This system included a modified tRNAPyl/Pyrrolysyl-tRNA synthetase (PylRS) pair from Methanosarcina mazei, the reporter gene D[TAG]G formed by DsRed and EGFP through amber stop codon TAG connection and the unnatural amino acid H-Lys(Boc)-OH. In silkworm BmE and BmNs cell lines, the reporter gene expression was strictly controlled by H-Lys(Boc)-OH in the presence of both PylRS and tRNAPyl. The silkworm genetic code expansion system established here is another useful controllable gene expression system besides tetracycline induced expression system.Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have established a genetic code expansion system for B. mori based on the well-researched tRNAPyl/PylRS pair from Methanosarcina mazei. We used H-Lys(Boc)-OH, which is a lysine derivative to efficiently and tightly control the expression of the reporter gene DsRed[TAG]EGFP (D[TAG]G), which encoded a H-Lys(Boc)-OH-bearing protein fused with DsRed and EGFP (here regarded as D[Boc]G) in B. mori cell lines BmE and BmNs. In D[TAG]G, the amber stop codon is recognized as the orthogonal tRNAPyl. Successful application of genetic code expansion system in silkworm cell lines will support the research into the function of silkworm genes and paves the way for the identification of new genes and protein markers in silkworm.

In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells.

Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system

BackgroundRice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria.Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants.ResultsWe introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants.ConclusionWe demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

Pinaverium Bromide Attenuates Lipopolysaccharide-Induced Excessive Systemic Inflammation via Inhibiting Neutrophil Priming

Abstract Dominant infiltration of neutrophils is a hallmark of many inflammatory diseases, especially in septic shock. IL-1β as one of the most early released proinflammatory cytokine in neutrophil, plays a pivotal role in the progress of sepsis. In this study, we built a high-throughput–compatible drug screen assay platform based on our newly constructed reporter C57BL/6 mice, pIL1-DsRed, expressing the DsRed gene under the control of the IL-1β promoter. After screening 1200 U.S. Food and Drug Administration–approved compounds, we found that pinaverium bromide (PVB) significantly suppressed the DsRed expression of primed neutrophil and improved the survival rate of mice given LPS in an endotoxin challenge analogous to sepsis, regardless of whether PVB was administered before or after LPS. PVB also protected the liver and lung from LPS-induced damage and reduced organ-specific inflammatory responses. PVB decreased the production of IL-1β, IL-6, and CXCL1 mRNA in the lungs of LPS-treated mice and decreased the serum levels of liver transaminases (alanine aminotransferase and aspartate aminotransferase) at multiple time points and doses tested. PVB can significantly suppress primed neutrophil-specific respiratory bursts and migration as well. Lastly, PVB affected neutrophils’ gene expression and phenotypic changes during neutrophil priming. PVB downregulated GM-CSF–induced expression of CD54 and dectin-2 (markers of fully primed neutrophils) at both mRNA and protein levels during late-phase neutrophil priming. In summary, we demonstrated that PVB can be used as a potential therapeutic agent for sepsis by inhibiting neutrophil priming.

Seeing is believing: a visualization toolbox to enhance selection efficiency in maize genome editing.

The emerging and promising biotechnology methodology of CRISPR/Cas9 gene-editing is revolutionizing crop improvement. However, the low-efficiency, time-consuming, and labor-intensive nature of positive or/and negative selections during or after transformation and subsequent identification of mutations are major challenges for its agricultural application, from upstream (high-throughput) mutant screening to downstream commercial production (He and Zhao, 2020). Although visual markers, and in particular fluorescent markers including green fluorescent protein (GFP) and red fluorescent protein (RFP), have been employed for the rapid visualization of transgenic material (Qi et al., 2020), the integration of cost-effective and non-invasive CRISPR-separation gene-editing tools is still in its infancy but remains critical for crop genetics and breeding (Callaway, 2018). Furthermore, special light sources are required to visualize fluorescent signals, which increases the cost and inconvenience of the application of fluorescence markers, especially in field conditions. We have developed a visualization toolbox, ViMeBox (VIsual Maize Editing toolBox), for the selection of positive transformants in maize (Zea mays). In the ViMeBox system, the vector expressing Cas9 includes a gene cassette containing a visible marker expressed from a tissue-specific promoter; Cas9-free kernels that are easy to separate by the visible marker are also undergoing gene editing. ViMeBox offers two advantages: 1) It enables enhanced expression of DsRED2, which makes seeds containing Cas9 visible to the naked eye in natural light, and does not affect genome editing efficiency or plant development. 2) It is effective for different seed tissues, for example, using embryo-specific promoters or promoters preferentially expressed in the embryo or aleurone. Furthermore, ViMeBox has potential applications in diverse additional scenarios.

Response of the porcine MYH4-promoter and MYH4-expressing myotubes to known anabolic and catabolic agents in vitro

Myosin heavy chain-IIB (MyHC-IIB; encoded by MYH4 or Myh4) expression is often associated with muscle hypertrophic growth. Unlike other large mammals, domestic pig breeds express MyHC-IIB at both the mRNA and protein level. AimTo utilise a fluorescence-based promoter-reporter system to test the influence of anabolic and catabolic agents on increasing porcine MYH4-promoter activity and determine whether cell hypertrophy was subsequently induced. MethodsC2C12 myoblasts were co-transfected with porcine MYH4-promoter-driven ZsGreen and CMV-driven DsRed expression plasmids. At the onset of differentiation, treatments (dibutyryl cyclic-AMP (dbcAMP), Des(1–3) Insulin-Like Growth Factor-1 (IGF-I), triiodo-l-thyronine (T3) and dexamethasone (Dex)) or appropriate vehicle controls were added and cells maintained for up to four days. At day 4 of differentiation, measurements were collected for total fluorescence and average myotube diameter, as indicators of MYH4-promoter activity and cell hypertrophy respectively. ResultsPorcine MYH4-promoter activity increased during C2C12 myogenic differentiation, with a marked increase between days 3 and 4. MYH4-promoter activity was further increased following four days of dbcAMP treatment and average myotube diameter was significantly increased by dbcAMP. Porcine MYH4-promoter activity also tended to be increased by T3 treatment, but there were no effects of Des(1–3) IGF-I or Dex treatment, whereas average myotube diameter was increased by Des(1–3) IGF-I, but not T3 or Dex. ConclusionPorcine MYH4-promoter activity responded to dbcAMP, Des(1–3) IGF-I and T3 treatment in vitro as observed previously in reported in vivo studies. However, we report that increased MYH4-promoter activity was not always associated with muscle cell hypertrophy. The fluorescence-based reporter system offers a useful tool to study muscle cell hypertrophic growth.

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase.

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.

ROS Regulate Caspase-Dependent Cell Delamination without Apoptosis in the Drosophila Pupal Notum.

SummaryThorax fusion occurs in the midline of the Drosophila pupal notum and involves epithelial cell delamination requiring apoptotic signaling. By genetic screening, we found that NADPH oxidases (Nox and Duox) associated with superoxide anion (O˙-2) are responsible for caspase-3 activation and delamination. We observed that Nox is upregulated in cells that undergo delamination and that delamination depends on caspase activation. However, the cell morphology and the almost complete lack of propidium iodide incorporation suggested little membrane disruption and signified apoptotic modulation. These results demonstrate that most delaminating cells undergo caspase activation, but this activation is not sufficient for apoptosis. We showed that the expression of Catalase, encoding an H2O2 scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of O˙-2 and intracellular H2O2 for delamination differs before and after caspase-3 activation, which involves live cell delamination.

Heterologous phytase expression in the food filamentous fungus <i> Aspergillus oryzae </i>using the added rice husk cultivation model

Aspergillus oryzae, a safe filamentous fungus, is widely used in food and enzyme production. In this study, we examined a cultivation model using rice husks as carrier to assess the capacity of recombinant protein production in A. oryzae. The model was first tested with the A. oryzae strain expressing the DsRed reporter gene. Expression of DsRed was easily detected by the pink color of the fungal mycelium on culture media and under a fluorescence microscope. The model was then evaluated with the phyA gene encoding a phytase from the fungus Aspergillus fumigatus. The phyA expression cassette regulated by the amyB promoter was permanently integrated into the genome of A. oryzae via Agrobacterium tumefaciens-mediated transformation with the pyrG nutritional marker. Results showed that A. oryzae transgenic strains carrying 2−3 copies of the phyA gene in their genomes exhibited a significant increase in phytase activity on agar medium supplemented with phytate. With rice husks added, these transgenic strains could secrete the recombinant phytase into the culture and phytase activity of the crude enzyme solution increased by 4.3 times compared to the unstransgenic A. oryzae. The established cultivation model and the transgenic approach in this study represent a potential for being used in production of secreted recombinant enzymes for animal feeds.

Effects of Certain cis-Regulatory Elements on Stage-Specific vitellogenin Expression in the Bombyx mori (Lepidoptera: Bombycidae)

Bombyx mori vitellogenin (BmVg) is highly upregulated during pupation, and the 20-hydroxyecdysone and amino acids may regulate stage-specific BmVg expression. However, previous studies showed that other factors may also affect stage-specific BmVg expression. Here, we characterized effective BmVg transcription factors by identifying the corresponding cis-regulatory elements (CREs). We prepared transgenic B. mori, in which DsRed was driven by various lengths of BmVg promoter. qRT-PCR analysis showed that DsRed expression driven by a 1.0-kb BmVg promoter (VgP1.0K) was consistent with endogenous BmVg. VgP1.0K specificity was closer to the endogenous BmVg promoter than that of VgP0.8K. These results suggest that CREs affecting stage-specific BmVg expression were localized to the 1.0-kb BmVg promoter. We investigated the effects of certain CREs that could influence the stage specificity of BmVg promoter on BmVg expression in transgenic B. mori. The relative DsRed expression was significantly reduced in transgenic female B. mori and the peak in DsRed expression was delayed after E-box CRE mutation. These results demonstrate that the E-box element enhanced BmVg expression and also affected stage-specific BmVg expression. Moreover, the relative DsRed expression was significantly increased in transgenic female of B. mori after 3×BD CRE mutation in BmVg promoter. However, the stage specificity of the mutated promoter was consistent with that of the endogenous BmVg promoter. The 3×BD element downregulated BmVg but had no effect on stage-specific BmVg expression. The present study promoted the process of elucidating the regulatory network for stage-specific BmVg expression and furnished a theoretical basis for the application of BmVg promoter.

Subcellular Localization of Rice Acyl-CoA-Binding Proteins ACBP4 and ACBP5 Supports Their Non-redundant Roles in Lipid Metabolism.

Acyl-CoA-binding proteins (ACBPs), conserved at the acyl-CoA-binding domain, can bind acyl-CoA esters as well as transport them intracellularly. Six ACBPs co-exist in each model plant, dicot Arabidopsis thaliana (thale cress) and monocot Oryza sativa (rice). Although Arabidopsis ACBPs have been studied extensively, less is known about the rice ACBPs. OsACBP4 is highly induced by salt treatment, but down-regulated following pathogen infection, while OsACBP5 is up-regulated by both wounding and pathogen treatment. Their differential expression patterns under various stress treatments suggest that they may possess non-redundant functions. When expressed from the CaMV35S promoter, OsACBP4 and OsACBP5 were subcellularly localized to different endoplasmic reticulum (ER) domains in transgenic Arabidopsis. As these plants were not stress-treated, it remains to be determined if OsACBP subcellular localization would change following treatment. Given that the subcellular localization of proteins may not be reliable if not expressed in the native plant, this study addresses OsACBP4:GFP and OsACBP5:DsRED expression from their native promoters to verify their subcellular localization in transgenic rice. The results indicated that OsACBP4:GFP was targeted to the plasma membrane besides the ER, while OsACBP5:DsRED was localized at the apoplast, in contrast to their only localization at the ER in transgenic Arabidopsis. Differences in tagged-protein localization in transgenic Arabidopsis and rice imply that protein subcellular localization studies are best investigated in the native plant. Likely, initial targeting to the ER in a non-native plant could not be followed up properly to the final destination(s) unless it occurred in the native plant. Also, monocot (rice) protein targeting may not be optimally processed in a transgenic dicot (Arabidopsis), perhaps arising from the different processing systems for routing between them. Furthermore, changes in the subcellular localization of OsACBP4:GFP and OsACBP5:DsRED were not detectable following salt and pathogen treatment, respectively. These results suggest that OsACBP4 is likely involved in the intracellular shuttling of acyl-CoA esters and/or other lipids between the plasma membrane and the ER, while OsACBP5 appears to participate in the extracellular transport of acyl-CoA esters and/or other lipids, suggesting that they are non-redundant proteins in lipid trafficking.

Development of a minigenome cassette for Lettuce necrotic yellows virus: A first step in rescuing a plant cytorhabdovirus.

Rhabdoviruses are enveloped negative-sense RNA viruses that have numerous biotechnological applications. However, recovering plant rhabdoviruses from cDNA remains difficult due to technical difficulties such as the need for concurrent in planta expression of the viral genome together with the viral nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L) and viral genome instability in E. coli. Here, we developed a negative-sense minigenome cassette for Lettuce necrotic yellows virus (LNYV). We introduced introns into the unstable viral ORF and employed Agrobacterium tumefaciens to co-infiltrate Nicotiana with the genes for the N, P, and L proteins together with the minigenome cassette. The minigenome cassette included the Discosoma sp. red fluorescent protein gene (DsRed) cloned in the negative-sense between the viral trailer and leader sequences which were placed between hammerhead and hepatitis delta ribozymes. In planta DsRed expression was demonstrated by western blotting while the appropriate splicing of introduced introns was confirmed by sequencing of RT-PCR product.

Engineering the cbh1 Promoter of Trichoderma reesei for Enhanced Protein Production by Replacing the Binding Sites of a Transcription Repressor ACE1 to Those of the Activators.

The strong and inducible cbh1 promoter is most widely used to express heterologous proteins, useful in food and feed industries, in Trichoderma reesei. Enhancing its ability to direct transcription provides a general strategy to improve protein production in T. reesei. The cbh1 promoter was engineered by replacing eight binding sites of the transcription repressor ACE1 to those of the activators ACE2, Hap2/3/5, and Xyr1. While changing ACE1 to Hap2/3/5-binding sites completely abolished the transcription ability, replacements with ACE2- and Xyr1-binding sites (designated cbh1pA and cbh1pX promoters, respectively) largely improved the promoter transcription efficiency, as reflected by expression of a reporter gene DsRed. The cbh1pA and cbh1pX promoters were applied to improve secretory expression of a codon-optimized mannanase from Aspergillus niger to 3.6- and 5.0-fold higher, respectively, which has high application potential in feed industry.

The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds in vivo

ABSTRACTVascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-Axxx and anti-angiogenic, anti-permeability VEGF-Axxxb. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-Axxx) and eGFP expression denotes distal splice site selection (VEGF-Axxxb). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-Axxx and VEGF-Axxxb. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo