Application Of Gene Silencing Research Articles

Construction and optimization of the TRV-mediated VIGS system in Areca catechu embryoids

Areca catechu, a member of the palm family Arecaceae native to tropical regions, holds significant economic importance as a tropical crop. This study explores the application of virus-induced gene silencing (VIGS) in A. catechu embryogenic callus tissues, utilizing the phytoene desaturase gene (AcPDS) of A. catechu as a reporter gene. Results demonstrate that employing EHA105 strain, an Agrobacterium infection liquid concentration of OD600 = 0.5, an infection duration of 5 min, acetosyringone (AS) concentration of 21.5 mg/L, co-cultivation at 19 °C for 2 days followed by 28 °C for 3 days, and a subsequent 14-day post-co-cultivation period, the experimental group (pTRV1+pTRV2-AcPDS) exhibited significantly reduced gene expression compared to the control group CK (pTRV1+pTRV2), manifesting the anticipated photobleaching phenotype by the 21st day. RT-PCR analysis revealed that AcPDS gene expression in TRV-mediated photobleached A. catechu embryos decreased to 0.227 times that of the control group CK. This established VIGS system in A. catechu embryoids establishes an effective experimental platform for studying functional genes in A. catechu.

Recent advances in virulence of a broad host range plant pathogen Sclerotinia sclerotiorum: a mini-review.

Sclerotinia sclerotiorum is a typical necrotrophic plant pathogenic fungus, which has a wide host range and can cause a variety of diseases, leading to serious loss of agricultural production around the world. It is difficult to control and completely eliminate the characteristics, chemical control methods is not ideal. Therefore, it is very important to know the pathogenic mechanism of S. sclerotiorum for improving host living environment, relieving agricultural pressure and promoting economic development. In this paper, the life cycle of S. sclerotiorum is introduced to understand the whole process of S. sclerotiorum infection. Through the analysis of the pathogenic mechanism, this paper summarized the reported content, mainly focused on the oxalic acid, cell wall degrading enzyme and effector protein in the process of infection and its mechanism. Besides, recent studies reported virulence-related genes in S. sclerotiorum have been summarized in the paper. According to analysis, those genes were related to the growth and development of the hypha and appressorium, the signaling and regulatory factors of S. sclerotiorum and so on, to further influence the ability to infect the host critically. The application of host-induced gene silencing (HIGS)is considered as a potential effective tool to control various fungi in crops, which provides an important reference for the study of pathogenesis and green control of S. sclerotiorum.

Current Perspectives of Matrix Metalloproteinase 11 (MMP11) as a Diagnostic and Therapeutic Target for Cancer

Abstract: Matrix metalloproteinase 11 (MMP11), also known as stromelysin-3, is a member of the matrix metalloproteinases family of proteins that are involved in physiological and pathological extracellular matrix remodelling. MMP11 does not hydrolyse classical MMP substrates, such as laminin and fibronectin, and many of its substrates remain unknown, piquing the interest of researchers. Several studies have reported the role of MMP11 in inducing tumour growth by inhibiting apoptosis and promoting cancer cell migration and invasion. Various reports have shown its potential as a diagnostic and prognostic marker in a majority of cancers. MMP11 also induces an immune response as a tumour-associated antigen, and recent evidence shows the involvement of many microRNAs in targeting MMP11 in cancer, with prospective future applications in cancer immunotherapy and gene silencing. Owing to the importance of MMP11 in both cancer diagnosis and therapy, there is a need for deeper understanding of its mechanism and role in tumour progression. The current review focuses on the role of MMP11 in cell signalling pathways, its expression status in various cancers, and its potential in cancer treatment.

Direct Conjugation of Gallium-(III)-Corroles to Short Interfering RNA(siRNA) Providing Real-Time siRNA Imaging and Gene Silencing.

Discovering new modifications for oligonucleotide therapeutics is essential for expanding its application to new targets and diseases. In this project, we focus on conjugating metaled ligands to short interfering RNAs (siRNAs) to investigate robust and simple conjugation methods for adding new properties such as real-time imaging to the siRNA. Here we report the chemical synthesis of novel Ga-(III)-corroles for their direct conjugation to siRNAs. Ga-(III)-corrole-siRNAs showed promising results when evaluated for gene silencing and live cell imaging. The knockdown activity of the firefly luciferase reporter gene was measured to evaluate gene silencing activity. Gene silencing studies from two 5'-Ga-(III)-labeled-siRNAs exhibited dose-dependent knockdown with IC50s of 812.7 and 451.4 pM, which is comparable to wild-type (IC50=439.7 pM) in the absence of red light, and IC50s of 562.9 and 354.5 pM, which is also comparable to wild-type (IC50=337.4 pM), in the presence of red light. In addition, imaging studies with Ga-(III)-corrole-modified siRNAs showed intense fluorescence in HeLa cells, highlighting that the Ga-(III)-corrole modification is an effective fluorophore for siRNA tracing and imaging. Moreover, the photodynamic activity of free base corrole vs the Ga-(III)-corrole was evaluated. Results show an increase of light cytotoxicity of the corrole ligand upon the addition of Ga-(III); however, no phototoxicity was observed when Ga-(III) ligands were linked to siRNA. In conclusion, Ga-(III)-corrole-siRNAs show promising results for applications in simultaneous real-time imaging and gene silencing.

Expanding the Toolbox for Genetic Manipulation in Pseudogymnoascus: RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in P. verrucosus.

Fungi belonging to the genus Pseudogymnoascus have garnered increasing attention in recent years. One of the members of the genus, P. destructans, has been identified as the causal agent of a severe bat disease. Simultaneously, the knowledge of Pseudogymnoascus species has expanded, in parallel with the increased availability of genome sequences. Moreover, Pseudogymnoascus exhibits great potential as a producer of specialized metabolites, displaying a diverse array of biological activities. Despite these significant advancements, the genetic landscape of Pseudogymnoascus remains largely unexplored due to the scarcity of suitable molecular tools for genetic manipulation. In this study, we successfully implemented RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption in Pseudogymnoascus, using an Antarctic strain of Pseudogymnoascus verrucosus as a model. Both methods were applied to target azpA, a gene involved in red pigment biosynthesis. Silencing of the azpA gene to levels of 90% or higher eliminated red pigment production, resulting in transformants exhibiting a white phenotype. On the other hand, the CRISPR/Cas9 system led to a high percentage (73%) of transformants with a one-nucleotide insertion, thereby inactivating azpA and abolishing red pigment production, resulting in a white phenotype. The successful application of RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption represents a significant advancement in Pseudogymnoascus research, opening avenues for comprehensive functional genetic investigations within this underexplored fungal genus.

Applications of Virus-Induced Gene Silencing in Cotton.

Virus-induced gene silencing (VIGS) is an RNA-mediated reverse genetics technique that has become an effective tool to investigate gene function in plants. Cotton is one of the most important economic crops globally. In the past decade, VIGS has been successfully applied in cotton functional genomic studies, including those examining abiotic and biotic stress responses and vegetative and reproductive development. This article summarizes the traditional vectors used in the cotton VIGS system, the visible markers used for endogenous gene silencing, the applications of VIGS in cotton functional genomics, and the limitations of VIGS and how they can be addressed in cotton.

Inducible Expression of dsRNA in Escherichia coli.

Double-stranded RNA (dsRNA) is a valuable tool for reverse genetics research and gene silencing applications. It is also an important management method for pests and diseases in agriculture. It can be synthesized both in vivo and in vitro. The latter presents the drawback of high production cost, the former is less expensive and suitable for scalable production. In general, dsRNAs are obtained in vivo from Escherichia coli heterologous systems that require the IPTG-inducible T7 RNA polymerase. In this report, we describe the construction of an RNAi system for the expression of dsRNA using the HT115 bacterial strain and the L4440 plasmid, and the extraction and identification of dsRNA.

Impact of the Core Chemistry of Self‐Assembled Spherical Nucleic Acids on their In Vitro Fate

AbstractNucleic acid therapeutics (NATs), such as mRNA, small interfering RNA or antisense oligonucleotides are extremely efficient tools to modulate gene expression and tackle otherwise undruggable diseases. Spherical nucleic acids (SNAs) can efficiently deliver small NATs to cells while protecting their payload from nucleases, and have improved biodistribution and muted immune activation. Self‐assembled SNAs have emerged as nanostructures made from a single DNA‐polymer conjugate with similar favorable properties as well as small molecule encapsulation. However, because they maintain their structure by non‐covalent interactions, they might suffer from disassembly in biologically relevant conditions, especially with regard to their interaction with serum proteins. Here, we report a systematic study of the factors that govern the fate of self‐assembled SNAs. Varying the core chemistry and using stimuli‐responsive disulfide crosslinking, we show that extracellular stability upon binding with serum proteins is important for recognition by membrane receptors, triggering cellular uptake. At the same time, intracellular dissociation is required for efficient therapeutic release. Disulfide‐crosslinked SNAs combine these two properties and result in efficient and non‐toxic unaided gene silencing therapeutics. We anticipate these investigations will help the translation of promising self‐assembled structures towards in vivo gene silencing applications.

Development and application of virus-induced gene silencing (VIGS) for studying ApTT8 gene function in Agapanthus praecox ssp. orientalis

Agapanthus praecox ssp. orientalis is a famous summer-blooming perennial that produces hundreds of florets with blue or white tepal. In comparison, other colours are infrequent in the genus Agapanthus. Previous analysis revealed that delphinidin was the main but not the only anthocyanidin, petunidin and cyanidin also existed in the tepal. This indicated that the colouration and regulation mechanism in A. praecox was unique and worthy of study. However, the stable genetic transformation was low efficiency and time-consuming. A rapid and effective gene functional identification method was urgently needed. In this study, the first use of the VIGS system in A. praecox was described in detail. Tobacco rattle virus (TRV) caused systemic infection with mild symptoms in A. praecox, which was also confirmed by qRT-PCR. The reporter gene ApPDS (phytoene desaturase) was silenced using the injection and soaking method in basal plates of inflorescence, and the photo-bleaching symptoms were observed in tepal. The optimal Agrobacterium concentration was OD600 = 1.0. Then the VIGS system was used to silence a bHLH-like transcriptional factor, ApTT8, to identify its function in anthocyanin biosynthesis. The results showed that the total anthocyanin content was reduced to 12.21-fold compared with the empty control. Additionally, tepals were taken as recipients of VIGS using vacuum infiltration method. Total anthocyanin content was reduced to 3.44-fold in ApTT8-silenced tepal. The silencing efficiency of tepal was as high as 85.09 %. The structure genes, ApF3’H, ApF3’5’H, ApF3H, ApDFR, ApANS and ApUFGT were also significantly down-regulated. The successful silencing of the ApPDS and ApTT8 in this study indicated that the optimized TRV-mediated VIGS system could be used to target gene silencing in florets and tepal of A. praecox. This method will significantly shorten the time required for studying molecular mechanism of flower colouration and is of great significance for the research of A. praecox functional genomics.

Application and Expansion of Virus-Induced Gene Silencing for Functional Studies in Vegetables

Increased consumption of vegetables has been recommended worldwide as a part of a healthy diet; therefore, determining gene function among breeding materials is crucial for vegetable improvement to meet the sustainable development of new vegetable varieties. However, genetic transformation is time-consuming and laborious, which limits the exploration of gene function for various vegetable crops. Virus-Induced Gene Silencing (VIGS) can perform large-scale and rapid gene silencing in plants due to a reduction in the experimental period and its independence from the stable genetic transformation, providing an excellent opportunity for functional research. VIGS can accelerate model plant research and make it easier to analyze gene function and validation in vegetable crops. Moreover, with the advent of technologies such as virus-mediated heterologous protein expression and the development of CRISPR/Cas9 technology, virus-mediated genetic tools have ushered in a new era in genetics and crop improvement. This study summarizes recent achievements in VIGS and Virus-Induced Gene Editing (VIGE) in vegetables. We also identify several challenges in the current state of VIGS technology in vegetables, serving as a guide for future research.

Molecular Targets and Mechanisms of 6,7-Dihydroxy-2,4-dimethoxyphenanthrene from Chinese Yam Modulating NF-κB/COX-2 Signaling Pathway: The Application of Molecular Docking and Gene Silencing

Chinese yam (Dioscorea opposita) tuber has a significant effect of invigorating the intestine and improving the symptoms of long-term diarrhea according to the records of the Chinese Pharmacopoeia. Phenanthrene polyphenols from Chinese yam, with higher inhibition of cyclooxygenase-2 (COX-2) than anti-inflammatory drugs, are an important material basis in alleviating ulcerative colitis via nuclear factor kappa-B (NF-κB)/COX-2 pathway, based on our previous research. The present study further explored the target and molecular mechanisms of phenanthrenes' modulation of the NF-κB/COX-2 signaling pathway by means of molecular docking and gene silencing. Firstly, interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) expression of 6-hydroxy-2,4,7-trimethoxyphenanthrene (PC2)/6,7-dihydroxy-2,4-dimethoxyphe-nanthrene (PC4) were compared on TNF-α induced human colon adenocarcinoma (Caco-2) cells. Secondly, molecular docking and dynamics simulation were implemented for PC2/PC4 and COX-2. Finally, COX-2 silencing was performed on TNF-α induced Caco-2 cells to confirm the target of PC4 on NF-κB/COX-2 pathway. Lower expression of IL-8 and TNF-α in PC4 treated Caco-2 cells indicated that PC4 had stronger anti-inflammatory activity than PC2. The binding of PC4 and COX-2 was stronger due to the hydrogen bond between hydroxyl group and Tyr385. No significant differences were found in phosphorylation nuclear factor kappa-B inhibitor alpha (pIkBα), phosphorylation NF-κB (pNF-κB) and phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2) expression between control and PC4 group after silencing, while these protein expressions significantly decreased in PC4 group without silencing, which confirmed that COX-2 was the important target for PC4 in alleviating ulcerative colitis. These findings indicate that PC4 was supposed to have inhibited NF-κB pathway mediated inflammation via suppression of positive feedback targeting COX-2.

Current Scenario of Exogenously Induced RNAi for Lepidopteran Agricultural Pest Control: From dsRNA Design to Topical Application.

Invasive insects cost the global economy around USD 70 billion per year. Moreover, increasing agricultural insect pests raise concerns about global food security constraining and infestation rising after climate changes. Current agricultural pest management largely relies on plant breeding-with or without transgenes-and chemical pesticides. Both approaches face serious technological obsolescence in the field due to plant resistance breakdown or development of insecticide resistance. The need for new modes of action (MoA) for managing crop health is growing each year, driven by market demands to reduce economic losses and by consumer demand for phytosanitary measures. The disabling of pest genes through sequence-specific expression silencing is a promising tool in the development of environmentally-friendly and safe biopesticides. The specificity conferred by long dsRNA-base solutions helps minimize effects on off-target genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera through non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to innovative solutions. Considerations about the regulation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives have revealed a worthy effort to control Lepidoptera pests with this new mode of action, which provides more sustainable and reliable technologies for field management. New data on the genomics of this taxon may contribute to a future customized target gene portfolio. As a case study, we illustrate how dsRNA and associated methodologies could be applied to control an important lepidopteran coffee pest.

Application of virus-induced gene silencing in Pogostemon cablin advances our understanding of patchoulol and sesquiterpene metabolism

ABSTRACT In plant species, virus-induced gene silencing (VIGS) effectively enables the transient knock down of target genes for functional analysis. To overcome the bottleneck preventing the functional genomics and biological engineering of Pogostemon cablin (Blanco) Benth. an important medicinal and industrial plant with high-value natural products, the VIGS system was established to enable the fast and efficient functional characterisation of candidate genes. In our study, the effectiveness of phytoene desaturase (PDS) gene silencing by VIGS was monitored by the visual indications in P. cablin and the activation effect spanned several months. VIGS of the PatPTS gene, encoding patchoulol synthase, caused a decline in expression abundance, resulting in a significant decreasing accumulation of patchouli alcohol and other sesquiterpenes. Cosilencing of PatPDS and PatPTS in P. cablin also suppressed the biosynthesis of patchouli alcohol. Negative feedback of the MVA and MEP pathway genes was demonstrated in PatPTS-silenced plants.

Recent Developments and Strategies for the Application of Agrobacterium-Mediated Transformation of Apple Malus × domestica Borkh.

Genetic transformation has become an important tool in plant genome research over the last three decades. This applies not only to model plants such as Arabidopsis thaliana but also increasingly to cultivated plants, where the establishment of transformation methods could still pose many problems. One of such plants is the apple (Malus spp.), the most important fruit of the temperate climate zone. Although the genetic transformation of apple using Agrobacterium tumefaciens has been possible since 1989, only a few research groups worldwide have successfully applied this technology, and efficiency remains poor. Nevertheless, there have been some developments, especially in recent years, which allowed for the expansion of the toolbox of breeders and breeding researchers. This review article attempts to summarize recent developments in the Agrobacterium-mediated transformation strategies of apple. In addition to the use of different tissues and media for transformation, agroinfiltration, as well as pre-transformation with a Baby boom transcription factor are notable successes that have improved transformation efficiency in apple. Further, we highlight targeted gene silencing applications. Besides the classical strategies of RNAi-based silencing by stable transformation with hairpin gene constructs, optimized protocols for virus-induced gene silencing (VIGS) and artificial micro RNAs (amiRNAs) have emerged as powerful technologies for silencing genes of interest. Success has also been achieved in establishing methods for targeted genome editing (GE). For example, it was recently possible for the first time to generate a homohistont GE line into which a biallelic mutation was specifically inserted in a target gene. In addition to these methods, which are primarily aimed at increasing transformation efficiency, improving the precision of genetic modification and reducing the time required, methods are also discussed in which genetically modified plants are used for breeding purposes. In particular, the current state of the rapid crop cycle breeding system and its applications will be presented.

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications.

Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators—DPQ and DPPZ—and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.

A novel aptamer-based small RNA delivery platform and its application to cancer therapy

Major challenges such as nuclease degradation, rapid renal clearance, non-specific delivery, poor cellular uptake and inflammatory response have limited the clinical application of small RNA-mediated gene silencing. To overcome these challenges, we designed a novel targeting small RNA delivery platform comprising of three oligonucleotides: (1) a guide RNA sequence, (2) part of a passenger sequence linked to a DNA aptamer via a PEG linker, and (3) another passenger sequence conjugated to cholesterol, which assemble through complementary base pair annealing. Remarkably, in the presence of magnesium, this molecule self-assembled into a nanoparticle with a hydrophobic cholesterol core, hydrophilic RNA oligonucleotide shell and PEG-linked DNA aptamer flare. The nanoparticles conferred protection to the RNA oligonucleotides against nuclease degradation, which increased bioavailability, and reduced systemic inflammatory responses. The aptamer allowed targeted delivery of RNA therapeutics through cell-specific surface markers, and once inside the cell, the nanoparticles induced lysosomal leakage that released the RNA oligonucleotides into the cytosol to achieve gene silencing. We created a c-Kit-targeting miR-26a delivery particle that specifically accumulated in c-Kit+ breast cancer, significantly increased T cell recruitment, and inhibited tumor growth. Regression of large established tumors were achieved when the nanoparticle was used in combination with anti-CTLA-4 monoclonal antibody.

TRUE Gene Silencing.

TRUE gene silencing is an RNA-mediated gene expression control technology and is termed after tRNase ZL-utilizing efficacious gene silencing. In this review, I overview the potentiality of small guide RNA (sgRNA) for TRUE gene silencing as novel therapeutics. First, I describe the physiology of tRNase ZL and cellular small RNA, and then sgRNA and TRUE gene silencing. An endoribonuclease, tRNase ZL, which can efficiently remove a 3′ trailer from pre-tRNA, is thought to play the role in tRNA maturation in the nucleus and mitochondria. There exist various small RNAs including miRNA and fragments from tRNA and rRNA, which can function as sgRNA, in living cells, and human cells appear to be harnessing cytosolic tRNase ZL for gene regulation together with these small RNAs. By utilizing the property of tRNase ZL to recognize and cleave micro-pre-tRNA, a pre-tRNA-like or micro-pre-tRNA-like complex, as well as pre-tRNA, tRNase ZL can be made to cleave any target RNA at any desired site under the direction of an artificial sgRNA that binds a target RNA and forms the pre-tRNA-like or micro-pre-tRNA-like complex. This general RNA cleavage method underlies TRUE gene silencing. Various examples of the application of TRUE gene silencing are reviewed including the application to several human cancer cells in order to induce apoptosis. Lastly, I discuss the potentiality of sgRNA as novel therapeutics for multiple myeloma.

Arabinonucleic acids containing C5-propynyl modifications form stable hybrid duplexes with RNA that are efficiently degraded by E. coli RNase H

The promise of the antisense approach to treat a variety of diseases with oligonucleotides and solutions to challenges that have been encountered in their development is attributable to chemical modification of the nucleic acid scaffold. Herein, we describe preliminary data regarding the synthesis of a novel C5-propynyl-β-d-arabinouridine (araUP) phosphoramidite and its incorporation into oligonucleotides. Substitution of araUP in dT18 results in minor stabilization of duplexes formed with RNA when modifications are placed consecutively and a uniformly modified araUP 18-mer increases stability by 34 °C relative to DNA. The modified oligomer exhibits improved nuclease and serum stability when compared to DNA and duplexes formed between RNA and araUP oligonucleotides are substrates for E. coli RNase H. These preliminary results merit further investigation into C5-propynyl modified arabino nucleic acids for potential therapeutic gene silencing applications.

Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

Biological and Medical Applications of Calcium Phosphate Nanoparticles.

Calcium phosphate nanoparticles have a high biocompatibility and biodegradability due to their chemical similarity to human hard tissue, for example, bone and teeth. They can be used as efficient carriers for different kinds of biomolecules such as nucleic acids, proteins, peptides, antibodies, or drugs, which alone are not able to enter cells where their biological effect is required. They can be loaded with cargo molecules by incorporating them, unlike solid nanoparticles, and also by surface functionalization. This offers protection, for example, against nucleases, and the possibility for cell targeting. If such nanoparticles are functionalized with fluorescing dyes, they can be applied for imaging in vitro and in vivo. Synthesis, functionalization and cell uptake mechanisms of calcium phosphate nanoparticles are discussed together with applications in transfection, gene silencing, imaging, immunization, and bone substitution. Biodistribution data of calcium phosphate nanoparticles in vivo are reviewed.