Single Gene Construct Research Articles

Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation

Cotton is one of the world’s largest fiber-producing crops. It is also known as the important cash crop of Pakistan. Cotton crop products contribute to economy as cottonseed oil, animal feed and the fiber support the textile industries, thus, playing a significant role in uplifting the economy of a country. But over time, bollworm attacks had escalated, severely reducing the yield of cotton and damaging cotton production. Bt (Bacillus thuringiensis) cotton is being widely used nowadays due to its enhanced resistance against cotton bollworms which are responsible for reducing crop production leading to high economic loss. It greatly affects the economy by increasing the cost of production and protection. Using resistant strains of Bt cotton not only makes it easier to combat bollworm attacks but also reduces the substantial expense issues that farmers face. Bt has many toxic proteins among which the two crystal proteins, Cry1Ac and Cry2Ab, are used to develop resistance against bollworms in cotton. This study aimed to optimize the developing transgenic callus containing Cry1Ac and Cry2Ab genes in cotton. The dual gene construct (Cry1Ac + Cry2Ab) provides more resistance to the cotton crop as compared to the single gene construct against whom the most bollworms have developed resistance. The vector was inoculated into the hypocotyl segments and the transformation process was preceded by shifting the hypocotyls at different mediums Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation. The construct-containing vector was introduced into a standard cotton line. A molecular confirmation test was carried out utilizing PCR and gene-specific primers after the formation of the transgenic callus. As a result of the research an indigenous cotton line that expresses double Bt genes for insect resistance was developed.

Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

GmGLU1 and GmRR4 contribute to iron deficiency tolerance in soybean.

Iron deficiency chlorosis (IDC) is a form of abiotic stress that negatively impacts soybean yield. In a previous study, we demonstrated that the historical IDC quantitative trait locus (QTL) on soybean chromosome Gm03 was composed of four distinct linkage blocks, each containing candidate genes for IDC tolerance. Here, we take advantage of virus-induced gene silencing (VIGS) to validate the function of three high-priority candidate genes, each corresponding to a different linkage block in the Gm03 IDC QTL. We built three single-gene constructs to target GmGLU1 (GLUTAMATE SYNTHASE 1, Glyma.03G128300), GmRR4 (RESPONSE REGULATOR 4, Glyma.03G130000), and GmbHLH38 (beta Helix Loop Helix 38, Glyma.03G130400 and Glyma.03G130600). Given the polygenic nature of the iron stress tolerance trait, we also silenced the genes in combination. We built two constructs targeting GmRR4+GmGLU1 and GmbHLH38+GmGLU1. All constructs were tested on the iron-efficient soybean genotype Clark grown in iron-sufficient conditions. We observed significant decreases in soil plant analysis development (SPAD) measurements using the GmGLU1 construct and both double constructs, with potential additive effects in the GmRR4+GmGLU1 construct. Whole genome expression analyses (RNA-seq) revealed a wide range of affected processes including known iron stress responses, defense and hormone signaling, photosynthesis, and cell wall structure. These findings highlight the importance of GmGLU1 in soybean iron stress responses and provide evidence that IDC is truly a polygenic trait, with multiple genes within the QTL contributing to IDC tolerance. Finally, we conducted BLAST analyses to demonstrate that the Gm03 IDC QTL is syntenic across a broad range of plant species.

On the road to C4 rice: advances and perspectives.

SummaryThe international C4 rice consortium aims to introduce into rice a high capacity photosynthetic mechanism, the C4 pathway, to increase yield. The C4 pathway is characterised by a complex combination of biochemical and anatomical specialisation that ensures high CO2 partial pressure at RuBisCO sites in bundle sheath (BS) cells. Here we report an update of the progress of the C4 rice project. Since its inception in 2008 there has been an exponential growth in synthetic biology and molecular tools. Golden Gate cloning and synthetic promoter systems have facilitated gene building block approaches allowing multiple enzymes and metabolite transporters to be assembled and expressed from single gene constructs. Photosynthetic functionalisation of the BS in rice remains an important step and there has been some success overexpressing transcription factors in the cytokinin signalling network which influence chloroplast volume. The C4 rice project has rejuvenated the research interest in C4 photosynthesis. Comparative anatomical studies now point to critical features essential for the design. So far little attention has been paid to the energetics. C4 photosynthesis has a greater ATP requirement, which is met by increased cyclic electron transport in BS cells. We hypothesise that changes in energy statues may drive this increased capacity for cyclic electron flow without the need for further modification. Although increasing vein density will ultimately be necessary for high efficiency C4 rice, our modelling shows that small amounts of C4 photosynthesis introduced around existing veins could already provide benefits of increased photosynthesis on the road to C4 rice.

Abstract PR08: Combinatorial CRISPR-Cas9 reveals many cancer synthetic lethal interactions are private to cell type

Abstract We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbation coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with multiplexed guide RNAs in two cell lines, testing 23,652 combinations. Numerous therapeutically relevant interactions were identified, most private to one cell line. These patterns replicated with combinatorial drugs at 80% precision. Thus, cellular context will be critical to synthetic-lethal therapies. Here, we combined multiplex targeting with array-based oligonucleotide synthesis to create dual-gRNA libraries covering up to 105 defined gene pairs. We conducted genetic interaction screens by transducing the dual-gRNA lentiviral library into a population of cells stably expressing Cas9, maintaining these cells in exponential growth over the course of four weeks, then sampling the relative changes in gRNAs at days 3, 14, 21 and 28 post-transduction. To robustly quantify gene fitness and genetic interactions, we developed a computational analysis framework that integrates all samples across the multiple days of the experiment. Using this method we evaluated all pairwise gene knockout combinations among a panel of 73 genes divided between tumor-suppressor genes (TSG) and cancer-relevant drug targets (DT), a subset of which were also verified oncogenes. Experiments were performed in two cancer cell lines: HeLa, a cervical cancer cell line driven by Human Papilloma Virus (HPV); and A549, a lung cancer cell line driven by KRAS G12S mutation. With nine gRNA pairs per combination, the library comprised 23,652 double gene knockout constructs and 657 single gene constructs; testing two replicates in each cell line yielded a total of 94,608 unique tests of interaction. Measurements of gene fitness (fg) were well correlated between biological replicates in the same cell line; Pearson r=0.96, p = 4.2×(10)^(-40), as were the π scores for significant genetic interactions; r = 0.75 p = 7.7×(10)^(-12). Moreover, we observed a significant correlation between the number of genetic interactions identified for a gene and its single gene fitness (HeLa: r = 0.77, p = 3.4×(10)^(-10); A549: r = 0.45, p = 0.0018), suggesting that network hubs may have increased functional importance to cancer cells relative to genes with fewer interactions; such a relationship has been previously observed in model organisms but not before in humans5. Interestingly, we found that the genetic interactions identified from these data were remarkably different between cell lines. A total of 97 synthetic-lethal (negative) genetic interactions were identified in either HeLa or A549 cells. Of these, only 12 were identified in both cell lines, while the remaining 85 interactions were private to a cell line (HeLa: 40 of 52, A549: 45 of 57). We next sought to validate these findings, and in particular the discrepancies across cell lines. We selected ten pairs of DT genes for which a synthetic-lethal genetic interaction had been identified in only one of the two cell lines. Rather than simply reproduce the dual CRISPR knockout experiment (gene-gene interaction), our goal was to examine the viability of cells exposed to drugs inhibiting the corresponding gene products (drug-drug interaction). In total, drug-drug assays validated eight of ten interactions when tested in the cell line for which the interaction had been first observed by dual CRISPR (80% precision or positive predictive value). In contrast, when the same ten gene pairs were tested in the other cell line that had not been implicated by dual CRISPR knockout, only two showed a negative genetic interaction by drug-drug assay (80% negative predictive value). Thus, the differences in genetic interaction across cell lines seen by systematic CRISPR could be largely reproduced as drug-drug interactions in small-scale assays. In summary, we have introduced a combinatorial CRISPR-Cas9 genetic interaction mapping technology that successfully identifies many therapeutically-relevant genetic interactions in cancer and shows the great importance of cellular context on the architecture of the genetic interaction network. Recognizing that there will be great diversity in genetic interaction between different tumors it will be important to perform these studies across a large number of samples, which is enabled by the high-throughput method we have developed. Citation Format: John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Amanda Birmingham, Ana Bojorquez-Gomez, Jason Kreisberg, Trey Ideker, Prashant Mali. Combinatorial CRISPR-Cas9 reveals many cancer synthetic lethal interactions are private to cell type [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR08.

Co-overexpression of the Constitutively Active Form of OsbZIP46 and ABA-Activated Protein Kinase SAPK6 Improves Drought and Temperature Stress Resistance in Rice.

Drought is one of the major abiotic stresses threatening rice (Oryza sativa) production worldwide. Drought resistance is controlled by multiple genes, and therefore, a multi-gene genetic engineering strategy is theoretically useful for improving drought resistance. However, the experimental evidence for such a strategy is still lacking. In this study, a few drought-responsive genes from rice were assembled by a multiple-round site-specific assembly system, and the constructs were introduced into the rice cultivar KY131 via Agrobacterium-mediated transformation. The transgenic lines of the multi-gene and corresponding single-gene constructs were pre-evaluated for drought resistance. We found that the co-overexpression of two genes, encoding a constitutively active form of a bZIP transcription factor (OsbZIP46CA1) and a protein kinase (SAPK6) involved in the abscisic acid signaling pathway, showed significantly enhanced drought resistance compared with the single-gene transgenic lines and the negative transgenic plants. Single-copy lines of this bi-gene combination (named XL22) and the corresponding single-gene lines were further evaluated for drought resistance in the field using agronomical traits. The results showed that XL22 exhibited greater yield, biomass, spikelet number, and grain number under moderate drought stress conditions. The seedling survival rate of XL22 and the single-gene overexpressors after drought stress treatment also supported the drought resistance results. Furthermore, expression profiling by RNA-Seq revealed that many genes involved in the stress response were specifically up-regulated in the drought-treated XL22 lines and some of the stress-related genes activated in CA1-OE and SAPK6-OE were distinct, which could partially explain the different performances of these lines with respect to drought resistance. In addition, the XL22 seedlings showed improved tolerance to heat and cold stresses. Our results demonstrate that the multi-gene assembly in an appropriate combination may be a promising approach in the genetic improvement of drought resistance.

Self-Assembled Enzyme Nanoparticles for Carbon Dioxide Capture.

Enzyme-based processes have shown promise as a sustainable alternative to amine-based processes for carbon dioxide capture. In this work, we have engineered carbonic anhydrase nanoparticles that retain 98% of hydratase activity in comparison to their free counterparts. Carbonic anhydrase was fused with a self-assembling peptide that facilitates the noncovalent assembly of the particle and together were recombinantly expressed from a single gene construct in Escherichia coli. The purified enzymes, when subjected to a reduced pH, form 50-200 nm nanoparticles. The CO2 capture capability of enzyme nanoparticles was demonstrated at ambient (22 ± 2 °C) and higher (50 °C) temperatures, under which the nanoparticles maintain their assembled state. The carrier-free enzymatic nanoparticles demonstrated here offer a new approach to stabilize and reuse enzymes in a simple and cost-effective manner.

A transgenic embryonic sexing system for the Australian sheep blow fly Lucilia cuprina.

Genetic approaches, including the sterile insect technique (SIT), have previously been considered for control of the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. In an SIT program, females consume 50% of the diet but are ineffective as control agents and compete with females in the field for mating with sterile males, thereby decreasing the efficiency of the program. Consequently, transgenic sexing strains of L. cuprina were developed that produce 100% males when raised on diet that lacks tetracycline. However, as females die mostly at the pupal stage, rearing costs would not be significantly reduced. Here we report the development of transgenic embryonic sexing strains of L. cuprina. In these strains, the Lsbnk cellularization gene promoter drives high levels of expression of the tetracycline transactivator (tTA) in the early embryo. In the absence of tetracycline, tTA activates expression of the Lshid proapoptotic gene, leading to death of the embryo. Sex-specific RNA splicing of Lshid transcripts ensures that only female embryos die. Embryonic sexing strains were also made by combining the Lsbnk-tTA and tetO-Lshid components into a single gene construct, which will facilitate transfer of the technology to other major calliphorid livestock pests.

Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice

Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment.

Tandem combination of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) generates a more potent antifungal protein

Plant defensins are small (45 to 54 amino acids) positively charged antimicrobial peptides produced by the plant species, which can inhibit the growth of a broad range of fungi at micro-molar concentrations. These basic peptides share a common characteristic three-dimensional folding pattern with one α-helix and three β-sheets that are stabilized by eight disulfide-linked cysteine residues. Instead of using two single-gene constructs, it is beneficial when two effective genes are made into a single fusion gene with one promoter and terminator. In this approach, we have linked two plant defensins namely Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) genes by a linker peptide sequence (occurring in the seeds of Impatiens balsamina) and made into a single-fusion gene construct. We used pET-32a+ vector system to express Tfgd2-RsAFP2 fusion gene with hexahistidine tag in Escherichia coli BL21 (DE3) pLysS cells. Induction of these cells with 1 mM IPTG achieved expression of the fusion protein. The solubilized His6-tagged recombinant fusion protein was purified by immobilized-metal (Ni2+) affinity column chromatography. The final yield of the fusion protein was 500 ng/μL. This method produced biologically active recombinant His6-tagged fusion protein, which exhibited potent antifungal action towards the plant pathogenic fungi (Botrytis cinerea, Fusarium moniliforme, Fusarium oxysporum, Phaeoisariopsis personata and Rhizoctonia solani along with an oomycete pathogen Phytophthora parasitica var nicotianae) at lower concentrations under in vitro conditions. This strategy of combining activity of two defensin genes into a single-fusion gene will definitely be a promising utility for biotechnological applications.

Toxoplasma gondii cathepsin proteases are undeveloped prominent vaccine antigens against toxoplasmosis

BackgroundToxoplasma gondii, an obligate intracellular apicomplexan parasite, infects a wide range of warm-blooded animals including humans. T. gondii expresses five members of the C1 family of cysteine proteases, including cathepsin B-like (TgCPB) and cathepsin L-like (TgCPL) proteins. TgCPB is involved in ROP protein maturation and parasite invasion, whereas TgCPL contributes to proteolytic maturation of proTgM2AP and proTgMIC3. TgCPL is also associated with the residual body in the parasitophorous vacuole after cell division has occurred. Both of these proteases are potential therapeutic targets in T. gondii. The aim of this study was to investigate TgCPB and TgCPL for their potential as DNA vaccines against T. gondii.MethodsUsing bioinformatics approaches, we analyzed TgCPB and TgCPL proteins and identified several linear-B cell epitopes and potential Th-cell epitopes in them. Based on these results, we assembled two single-gene constructs (TgCPB and TgCPL) and a multi-gene construct (pTgCPB/TgCPL) with which to immunize BALB/c mice and test their effectiveness as DNA vaccines.ResultsTgCPB and TgCPL vaccines elicited strong humoral and cellular immune responses in mice, both of which were Th-1 cell mediated. In addition, all of the vaccines protected the mice against infection with virulent T. gondii RH tachyzoites, with the multi-gene vaccine (pTgCPB/TgCPL) providing the highest level of protection.ConclusionsT. gondii CPB and CPL proteases are strong candidates for development as novel DNA vaccines.

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons

The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as "vitamin E") as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we (i) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, (ii) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and (iii) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

Enhanced polyhydroxybutyrate production in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.

Human Sodium Iodide Symporter Added to Multidrug Resistance 1 Small Hairpin RNA in a Single Gene Construct Enhances the Therapeutic Effects of Radioiodine in a Nude Mouse Model of Multidrug Resistant Colon Cancer

The objective of this study was to investigate the therapeutic potential of ¹³¹I added to doxorubicin therapy in multidrug resistance (MDR) mouse colon cancer coexpressing the MDR1 small hairpin RNA (shRNA) and human sodium iodide symporter (hNIS) gene in a single gene construct and to visualize the antitumor effects using molecular nuclear imaging. HCT-15 coexpressing shRNA for MDR1 gene (MDR1 shRNA) and hNIS gene with a single construct was established (referred to as MN61 cell). Inhibition of P-gp function by MDR1 shRNA and functional activity of hNIS gene was assessed using a ⁹⁹(m)Tc sestamibi uptake and ¹²⁵I uptake, respectively. Cytotoxic effects by a combination of doxorubicin and ¹³¹I were determined in parental (HCT-15) or MN61 cells using an in vitro clonogenic assay. Therapeutic effect of either combination therapy (doxorubicin and ¹³¹I) or single therapy (doxorubicin or ¹³¹I alone) was evaluated by tumor volume measurement. ⁹⁹(m)Tc-sestamibi, ¹²³I, and ⁹⁹(m)Tc-pertechnetate images of mice were acquired to evaluate functional assessment in vivo. Cellular uptake of ⁹⁹(m)Tc-sestamibi and ¹²⁵I was approximately 2-fold and 100-fold higher in MN61 cells than in parental cells, respectively. Combination of ¹³¹I and doxorubicin resulted in higher cytotoxcity in MN61 cells as compared with parental cells. Scintigraphic imaging showed higher uptake of ⁹⁹(m)Tc-sestamibi and ¹²³I in MN61 tumor as compared with parental tumor. In mice treated with doxorubicin, there was a slight delay in tumor growth in the MN61 tumor but not in the parental tumor. Cancer treatment with ¹³¹I or doxorubicin induced a rapid reduction of tumor volume in the MN61 tumor but not in the parental tumor. Combination therapy further generated a rapid reduction of tumor volume as compared with ¹³¹I therapy alone (p < 0.05). A combination hNIS mediated radioiodine gene therapy added to MDR1 shRNA treatment improved the effects of cancer treatment in a MDR cancer model and could enable visualization of the antitumor effects with nuclear imaging.

Transgene Stacking and Coordinated Expression of Plant Defensins Confer Fungal Resistance in Rice

Abstract Transgenic rice with cleavable chimeric polyprotein and single-protein gene constructs placed under the control of single maize ubiquitin promoter were generated by Agrobacterium tumefaciens-mediated transformation. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), Dm-AMP1 and Rs-AFP2, from the seeds of Dahlia merckii and Raphanus sativus, respectively. These genes were linked by a 16 amino acid Ib-AMP linker peptide region isolated from the seeds of Impatiens balsamina. Average expression of Dm-AMP1 and Rs-AFP2 was 8.35 ± 0.56 and 8.1 ± 0.6 2 g/mg of total soluble proteins in the leaf extracts of transgenic plants. Plants transformed with polyprotein construct showed significantly improved disease resistance against Magnaporthe oryzae and Rhizoctonia solani by 90% and 79%, respectively, as compared to untransformed plants. The plants transformed with polyprotein construct were more resistant to M. oryzae compared to plants transformed with control single-gene constructs of Dm-AMP1 and Rs-AFP2. The chimeric polyprotein was cleaved and present in equimolar concentration in transgenic rice and the individual AMPs were secreted into the extracellular space. The results of in vitro, in planta, and microscopic analyses suggest that chimeric polyprotein expression has the potential to provide broad-spectrum disease resistance in rice.

Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley stripe mosaic virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes after wheat plants were infected with BSMV-VIGS constructs carrying 178bp fragments of the PDS and SGT1 genes linked in cis or after infection with single-gene silencing BSMV constructs carrying PDS or SGT1 alone or with a mixture of the single-gene constructs. Although significant variation occurred in these studies it was clearly demonstrated that two wheat genes can be silenced simultaneously by a single BSMV-VIGS construct.

Mass Spectrometry Reveals the Missing Links in the Assembly Pathway of the Bacterial 20 S Proteasome

The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 alpha-type and 14 beta-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (alpha7beta7beta7alpha7). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing short-lived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early alpha/beta-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the beta-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half- and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a beta-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.

Abstract 186: Biopacemakers Induced by Gene Transfer of a Dual-Functional Kir2.1 Mutant

Previous efforts to engineer biopacemakers have focused either on overexpression of HCN, or on suppression of I K1 to liberate endogenous pacemaker activity. Here we report a novel strategy designed to convert I K1 into a nonselective “leak” current. The idea is to combine the key virtues of HCN (leak current) and I K1 suppression (destabilization of the resting potential) in a single gene construct. We focused on the human Kir2.1ER mutation (E138R and R148E, KER) which can work as not only a functional non-selective channel in homotetramers but also as a dominant-negative suppressor for the Kir2.X gene family in heart. The plasmid CMV-KER-IRES-wtKir2.1 coexpresses the KER mutant and wild-type Kir2.1. In HEK cells transduced with CMV-KER-IRES-wtKir2.1, barium sensitive hyperpolarization-activated inward current (−5.5 pA/pF at −80mV) could be detected (n=5) with a reversal potential of −35.1± 2.1 mV in normal Tyrode external solution. These data demonstrate that overexpression of KER undermines the ability of Kir to produce functional I K1 channels, while producing a cation-nonselective leak current. To probe the consequences of KER in primary tissues, we created the adenovirus AdKER in which KER expression is driven by the CMV promoter. Isolated guinea-pig myocytes transduced with AdKER exhibited spontaneous action potential oscillations with high reproducibility (n=10). Electrocardiograms performed 3 days after AdKER injection into guinea pig hearts showed idioventricular rhythms in virus injected-animals, while no such rhythms were seen in control (AdGFP) animals (n=6 each). These results show that KER mutants, if expressed in ventricular muscle, produce biopacemakers in vitro and in vivo . This study provides proof of principle for the notion that biological pacemakers can be readily induced by a single gene designed to destabilize I K1 and to create a leak current.

Liposomal plasmid DNA encoding human thymosin α1 and interferon ω1 potently inhibits liver tumor growth in ICR mice

To evaluate the potential therapeutic effect of liposomal gene delivery, genes encoding for human thymosin alpha1 (Talpha1) and interferon omega1 were injected via the tail vein into mice bearing a Hep-A-22 liver tumor. The cDNA of human Talpha1 and interferon omega1 were obtained by synthesis or reverse transcription-polymerase chain reaction (RT-PCR), respectively. Eukaryotic expressing vectors pIRES2, encoding Talpha1 and/or interferon omega1, were constructed and injected with liposome via the tail vein into ICR mice bearing a Hep-A-22 tumor. The potency of tumor inhibition was evaluated when three treated groups were compared with the group receiving the empty vector. Apoptosis of tumor cells was investigated by analyzing DNA fragmentation. Only the group treated with dual-gene plasmid reached an eligible level of tumor inhibition (43%). The difference in tumor weight was statistically significant between the Talpha1 gene or the interferon omega1 gene treated groups and the control (P<0.05), and highly significant between the dual-gene treated group and the control (P<0.01). DNA ladder was observed in the tumor cells from the purpose gene treated groups but not from the control. The dual-gene plasmid-liposome complex showed more potent inhibition than the single gene constructs on the growth of Hep-A-22 tumor cells in mice, which may be attributed to indirect and additive induction of apoptosis in tumor cells by increased expression of Talpha1 and interferon omega1.

Simultaneous production of two foreign proteins from a potyvirus-based vector

With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial β-glucuronidase (GUS) gene ( uidA). Two insertion sites were assessed, either between P1 and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for P1 and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Western blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector.