Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24

Virus-induced gene silencing database for phenomics and functional genomics in Nicotiana benthamiana

Virus-induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built-in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword-based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org.Noble Research Institute and NSF IOS-102564

Internuclear gene silencing in Phytophthora infestans is established through chromatin remodelling

In the plant pathogen Phytophthora infestans, nuclear integration of inf1 transgenic DNA sequences results in internuclear gene silencing of inf1. Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in P. infestans. The DNA methylation inhibitor 5-azacytidine induced reversion of the inf1-silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse inf1 silencing. inf1-expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic inf1-silenced strains that were generated via internuclear gene silencing, where inf1 expression was restored permanently. Therefore, inf1-transgenic sequences are required to maintain the silenced state. Prolonged culture of non-transgenic inf1-silenced strains resulted in gradual reactivation of inf1 gene expression. Nuclease digestion of inf1-silenced and non-silenced nuclei showed that inf1 sequences in silenced nuclei were less rapidly degraded than non-silenced inf1 sequences. Bisulfite sequencing of the endogenous inf1 locus did not result in detection of any cytosine methylation. Our findings suggest that the inf1-silenced state is based on chromatin remodelling

A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies

We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae

Factors influencing Barley Stripe Mosaic Virus-mediated gene silencing in wheat

Virus induced gene silencing (VIGS) is a technology that has been used primarily to target the virus genome in infected plants. However, if the virus genome carries inserts derived from the host plant, the system could be employed to target the mRNAs corresponding to the host gene. Barley Stripe Mosaic Virus (BSMV), a disease of various cereals including barley and wheat, has been one of the successfully used viral RNA silencing tools in monocotyledonous plants for the last decade. In this study, we investigated several factors that play a significant role in VIGS. We tested the efficiency of silencing two genes simultaneously using the BSMV-induced gene silencing (IGS) system in detail. We found that two genes could be silenced simultaneously using BSMV-IGS. However, the silencing efficiency was found to be influenced by several factors including stability of the insert, temperature, and the accumulation of small viral RNAs from BSMV. The effect of these factors on VIGS system has been discussed

Ultrasound assisted siRNA delivery using PEG-siPlex loaded microbubbles

Short interfering RNA (siRNA) attracts much attention for the treatment of various diseases. However, its delivery, especially via systemic routes, remains a challenge. Indeed, naked siRNAs are rapidly degraded, while complexed siRNAs massively aggregate in the blood or are captured by macrophages. Although this can be circumvented by PEGylation, we found that PEGylation had a strong negative effect on the gene silencing efficiency of siRNA-liposome complexes (siPlexes). Recently, ultrasound combined with microbubbles has been used to deliver naked siRNA but the gene silencing efficiency is rather low and very high amounts of siRNA are required. To overcome the negative effects of PEGylation and to enhance the efficiency of ultrasound assisted siRNA delivery, we coupled PEGylated siPlexes (PEG-siPlexes) to microbubbles. Ultrasound radiation of these microbubbles resulted in massive release of unaltered PEG-siPlexes. Interestingly, PEG-siPlexes loaded on microbubbles were able to enter cells after exposure to ultrasound, in contrast to free PEG-siPlexes, which were not able to enter cells rapidly. Furthermore, these PEG-siPlex loaded microbubbles induced, in the presence of ultrasound, much higher gene silencing than free PEG-siPlexes. Additionally, the PEG-siPlex loaded microbubbles only silenced the expression of genes in the presence of ultrasound, which allows space and time controlled gene silencing

RNA-Mediated Gene Silencing Signals Are Not Graft Transmissible from the Rootstock to the Scion in Greenhouse-Grown Apple Plants Malus sp.

RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent

An Analysis of Putative Roles for the CCR4-NOT Deadenylase-Complex Subunit Regena (NOT2) in microRNA-Mediated Gene Silencing in \u3cem\u3eDrosophila Melanogaster\u3c/em\u3e

microRNAs (miRNAs) are one class of small non-coding ribonucleic acid (RNA) molecules essential to development and homeostasis in plants and animals. miRNAs silence gene expression through complementary base pairing with target gene messenger RNAs and association with the miRNA-induced silencing complex (miRISC). The identification and characterization of cellular factors required for miRNA-mediated gene silencing is incomplete. A forward genetic screen was carried out in Drosophila melanogaster to generate flies defective for gene silencing. Silencing was assayed by expression of a Green Fluorescent Protein (GFP) reporter fused to the Brd gene 3’ UTR, which is regulated by miRNAs. Genetic analysis revealed that the CCR4-NOT deadenylase-complex subunit Regena (NOT2) is required for miRNA-mediated silencing of the reporter. In addition, perturbation of Regena function altered Drosophila eye development and resulting adult eye morphology. miRNAs are thought to silence target gene expression through a combination of translational repression and target mRNA degradation, though the detailed mechanism of this process is a matter of controversy. Novel genetic reagents to explore miRNA function in vivo have been generated and characterized. Ongoing efforts aim to explore whether Regena is required to silence other miRNA targets in vivo, and whether Regena is required for miRNA-mediated gene silencing at different stages of the Drosophila life cycle. Elucidation of the lesion in the Regena (NOT2) gene and the molecular nature of GFP reporter silencing will contribute to an understanding of the mechanism of miRNA-mediated gene silencing in vivo

Intramyocardial gene silencing by interfering RNA

RNAi is a widely used methodology for gene silencing. The action mechanism of siRNA molecules has been well studiedin recent years, and the technique has been optimized in terms of safety and effectiveness. Cardiovascular diseases havea high incidence in the current population, and despite of the extensive research, safe and efficient therapeutics have notyet been found, which is reflected by 17.1 million people who die each year for this cause. In this context, siRNAs arebeing considered a therapeutic tool to regulate the expression of genes involved in the generation of these pathologies.The efficacy of siRNAs entry to cardiomyocytes, the safety of the delivery process and the degree of silencing achievedare main aspects before consider it as a cardiovascular disease therapy. Presently, we will give a brief outline of thecurrent understanding of the RNAi mechanism and the delivery system to the heart. We describe the use of lentivirus fora functional silencing of cardiac proteins in the study of a pathophysiological process, the slow force response to cardiacstretch.Fil: Brea, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Morgan, Patricio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Perez, Nestor Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; Argentin