Green Fluorescent Protein mRNA Research Articles

O17 Highly efficient base editing of homozygous and heterozygous COL7A1 variants in fibroblasts from patients with recessive dystrophic epidermolysis bullosa using adenine base editor 8e mRNA system delivered via novel lipid nanoparticle formulations

Abstract Introduction and aims Adenine base editors (ABEs) present an attractive approach to gene editing in the inherited blistering disease recessive dystrophic epidermolysis bullosa (RDEB), which Results from pathogenic variants in COL7A1. We previously showed that ABE8e, a new ABE variant, confers remarkable efficiency (94.6%) and a favourable safety profile in correcting variants and restoring collagen 7 (C7) expression when electroporated into RDEB fibroblasts. With a goal to develop topical base editing creams, we now explore use of lipid nanoparticle (LNP)-encapsulated ABE8e mRNA system to edit two distinct variants in RDEB fibroblasts. Methods Primary patient fibroblasts harbouring a homozygous G>A mutation (exon 42) or a heterozygous C>T mutation (exon 54) were gene edited using ABE8e mRNA, delivered via lipofectamine or six different LNPs. To assess the gene editing efficiency, Sanger sequencing and next-generation sequencing were employed. Cell viability was evaluated via a lactate dehydrogenase cytotoxicity assay, while functional restoration of C7 expression was assessed via Western blotting of cell lysate and supernatant. Finally, topical delivery was tested on normal human skin equivalents (HSEs). Results We identified an optimal ratio of 1 : 7 for sgRNA : ABE8e, at which editing efficiency increases with ABE8e dosage, with 400 ng being sufficient for 90% biallelic editing. Formulations containing DTDTMA (C14) and DHDTMA (C16) LNPs demonstrated approximately 90% and approximately 80% editing (with and without the peptide, respectively), in addition to reduced cytotoxicity compared with lipofectamine. Depending on the nature of the mutation, the edit corresponded to an increase in C7 expression in supernatant only or cell lysate as well, whereby C14 LNPs enabled twofold C7 restoration. Finally, our HSEs demonstrated viability of topical delivery of lipofectamine-encapsulated green fluorescent protein mRNA. Conclusions We demonstrated the potential value of base editing as a gene therapy for RDEB in addition to the potential of these LNPs for clinical translation of the therapy. Nonetheless, further safety and functionality tests still need to be conducted.

Escherichia coli methionine-tRNAi/methionyl tRNA synthetase pairs induced protein initiation of interest (PII) expression

The precise regulatory role in protein synthesis by facilitating interactions with mRNA codons for various tRNA modifications is unclear. We previously reported that enhanced green fluorescent protein (GFP) reduced enhanced GFP mRNA expression in human methionine-conjugated initiator tRNA (tRNAi)/tRNA synthetase pairs under methionine-deficient conditions. Here, we investigated the effect of non-formylated methionine-conjugated Escherichia coli tRNAi on the synthesis of the protein initiation of interest (PII) in HeLa cells under intracellular L-methionine levels. We found that E. coli methionine-tRNAi counteracts human methionine-tRNAi, indicating that E. coli methionyl tRNA synthetase can induce enhanced GFP expression due to increased stability of enhanced GFP mRNA. Both complexes could support translation initiation without being employed to introduce methionine residues in the subsequent elongation steps. The results indicated that E. coli methionine-tRNAi could offset human methionine-tRNAi, and E. coli methionine-tRNAi/methionyl tRNA synthetase pairs can drive enhanced GFP mRNA expression. Unlike the human methionine-tRNAi/methionyl tRNA synthetase pairs that were used as a positive control, the non-formylated E. coli methionine-tRNAi/methionyl tRNA synthetase pairs reduced the expression of enhanced GFP mRNA, resulting in reduced HeLa cell survival. Using tRNAs functions causes of heterologous origin, such as from prokaryotes, and modified, to enhance or suppress the synthesis of specific proteins in eukaryotic organisms into the potential may possess a more prominent advantage of E. coli methionine-tRNAi as approaches that can control PII. This study provides new insights on the E. coli methionine- tRNAi/methionyl tRNA synthetase pair induced PII synthesis and the relative viability of cells could pave the way to regulate ecological/biological systems.

Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways

Introduction: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19.Methods: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging.Results: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73–97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control.Conclusion: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

CRISPR/Cas9-mediated knockout of the DCL2 and DCL4 genes in Nicotiana benthamiana and its productivity of recombinant proteins.

DCL2 and DCL4 genes in Nicotiana benthamiana plants were successfully edited using the CRISPR/Cas9 system. Recently, plants have been utilized for recombinant protein production similar to other expression systems, i.e., bacteria, yeast, insect, and mammal cells. However, insufficient amounts of recombinant proteins are often produced in plant cells. The repression of RNA silencing within plant cells could improve production levels of recombinant protein because RNA silencing frequently decomposes mRNAs from transgenes. In this study, the genes dicer-like protein 2 and 4 (NbDCL2 and NbDCL4) were successfully edited to produce double-knockout transgenic Nicotiana benthamiana plants (dcl2dcl4 plants) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. A transient green fluorescent protein (GFP) gene expression assay revealed that the dcl2dcl4 plants accumulated higher amounts of GFP and GFP mRNA than wild type (WT) and RNA-dependent RNA polymerase 6-knockout N. benthamiana plants (ΔRDR6 plants). Small RNA sequencing also showed that dcl2dcl4 plants accumulated lower amounts of small interfering RNAs (siRNAs) against the GFP gene than WT plants. The dcl2dcl4 plants might also produce higher amounts of human fibroblast growth factor 1 (FGF1) than WT and ΔRDR6 plants. These observations appear to reflect differences between DCLs and RDR6 in plant cell biological mechanisms. These results reveal that dcl2dcl4 plants would be suitable as platform plants for recombinant protein production.

Controlled Differentiation of Mesenchymal Stem Cells into Hyaline Cartilage in miR-140-Activated Collagen Hydrogel.

Hypertrophic cartilage formation is a major setback in mesenchymal stem cells (MSCs)-mediated cartilage repair, and overcoming it requires optimization of differentiation. Here, we tested the miR-140 activated collagen hydrogel for the chondrogenic differentiation of MSCs and to produce hyaline cartilage. Bone marrow MSCs isolated from 3 patients were pretreated with miR-140 and then chondrogenic differentiated. The 3-dimensional (3D) transfection potential of 5 different transfection reagents (Polyethylenimine, Lipofectamine, TransIT-X2, Amide:Cholesterol-based liposomes [AmC] and AmC pegylated with Tocofersolan [AmCTOC]) was compared and the reagent that showed higher green fluorescent protein (GFP) expression was selected. Finally, the collagen hydrogel was activated using miR-140-transfection complex and sustained delivered to MSCs during chondrogenic differentiation. After differentiation, the outcome was assessed by reverse transcription-polymerase chain reaction (RT-PCR), histology, immunohistochemistry, and compared with scrambled miRNA treated control. Pretreatment of MSCs with miR-140 significantly increased the expression of cartilage-specific genes (COL2A1, SOX9, and ACAN) with reduced hypertrophic chondrocyte (COL10A1) marker expression and better safranin-O staining than the control. The AmCTOC liposome showed a significant increase in 3D transfection of GFP expressing plasmid than the others. Furthermore, the knockdown of GAPDH using siRNA in HEK cells and expression of GFP mRNA in human bone marrow MSCs confirmed the 3D-transfection efficiency of AmCTOC. The sustained delivery of miR-140 using activated matrix formed a hyaline cartilage-like tissue with minimal COL10A1 expression in RT-PCR and immunohistochemistry. Our results demonstrated the therapeutic potential of miR-140-activated hydrogel for MSCs-based cartilage tissue engineering, which could also be used for endogenous stem cells-mediated cartilage repair.

Identification of the optimal insertion site for expression of a foreign gene in an infectious hematopoietic necrosis virus vector.

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.

CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins.

RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.

Novel in vivo system to monitor tRNA expression based on the recovery of GFP fluorescence and its application for the determination of plant tRNA expression

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5′-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5′-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3′-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.

In-vitro Transcribed mRNA Delivery Using PLGA/PEI Nanoparticles into Human Monocyte-derived Dendritic Cells.

Induction of protein synthesis by the external delivery of in-vitro transcription-messenger RNA (IVT-mRNA) has been a useful approach in the realm of cell biology, disease treatment, ‎reprogramming of cells, and vaccine design. Therefore, the development of new formulations for ‎protection of mRNA against nucleases is required to maintain its activity in-vivo. It was the aim of the present study to ‎investigate the uptake, toxicity, transfection efficiency as well as phenotypic consequences of ‎a nanoparticle (NP) in cell culture. NP consists of poly D, L-lactide-co-glycolide (PLGA) and polyethyleneimine (PEI) ‎for delivery of in-vitro transcription-messenger RNA (IVT- mRNA) encoded green fluorescent protein (GFP) in human monocyte-derived ‎dendritic cells (moDCs). Nanoparticles that were synthesized and encapsulated with synthetic GFP mRNA, exhibited size distribution in this formulation, with mean particle sizes ranging between 415 and 615 nm. Zeta potential was positive (above 12-13 mV) and the encapsulation efficiency exceeded 73.5%. Our results demonstrated that PLGA/PEI NPs encapsulation of GFP mRNA had ‎no toxic effect on immature monocyte-derived ‎dendritic cells and was capable of delivering of IVT-mRNA into moDCs and was highly effective. The expression of GFP protein 48 h after transfection was confirmed by flow cytometry, microscopic examination and western blotting assay. This NP can make a way to target moDCs to express a variety of antigens by IVT- mRNA. The ‎present study introduced the PLGA/PEI NP, which provided effective delivery of ‎IVT-mRNA that encodes the GFP protein.

The V2 protein encoded by a monopartite begomovirus is a suppressor of both post-transcriptional and transcriptional gene silencing activity

Papaya leaf curl virus (PaLCuV) is a begomovirus (genus Begomovirus; family Geminiviridae) with a monopartite genome that is usually associated with beta- and alphasatellites in plants. Geminiviruses are DNA viruses with small circular genomes that occur as minichromosomes in the nucleus and are susceptible to post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). Transient expression of the PaLCuV V2 (PV2) protein together with the green fluorescent protein (GFP) in Nicotiana benthamiana resulted in enhanced levels of GFP fluorescence and GFP mRNA, indicative of suppression of PTGS. Expression of PV2 from a Potato virus X vector restored GFP expression in N. benthamiana plants harbouring a transcriptionally silenced GFP transgene, indicative of suppression of TGS. The results show that the PV2 protein encoded by PaLCuV has both suppressor of PTGS and TGS activity and is an important factor in overcoming host RNA-silencing based defenses.

Universal mRNA Translation Enhancement with Gold Nanoparticles Conjugated to Oligonucleotides with a Poly(T) Sequence.

DNA-conjugated gold nanoparticles (AuNPs) have been shown to enhance the translation of mRNA. However, the specific sequence on the DNA dictates the specific mRNA to be enhanced. This study describes poly(thymine)-functionalized AuNPs (AuNP-p(T)DNA) capable of enhancing the translation of any mRNA template that is incorporated into pcDNA6 vector with bovine growth hormone (BGH) polyadenylation signal (P(A)). We demonstrated this by incorporating four genes: green fluorescence protein (GFP), general control nonderepressible 5 (GCN5), cAMP-responsive element binding protein 1 (CREB1), and X-box-binding protein 1-spliced (XBP-1S) separately into pcDNA6 vector with BGH P(A) before their expression in HeLa lysate. The addition of AuNP-p(T)DNA to HeLa lysate containing GFP, GCN5, CREB1, and XBP-1S mRNA increased protein synthesis 1.80, 1.99, 1.95, and 2.20 times, respectively. Similar translation enhancement was also observed in a multiplex reaction containing the mRNA of three genes together in the lysate. Complementary p(T)DNA hybridization to the poly(A) tail of the mRNA was critical as the removal of either p(T)DNA or BGH P(A) in XBP-1S mRNA or the replacement of p(T)DNA with p(A)DNA reduced the translation back to baseline level. Finally, an optimum length of 25 nucleotides for the DNA oligomer and a AuNP-p(T)DNA:mRNA ratio of 0.658 achieved a 3.08-fold translation enhancement. The AuNP-p(T)DNA nanoconstruct could be incorporated into commercial cell-free protein synthesis kits as a universal translation enhancer.

The Rep proteins encoded by alphasatellites restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

Alphasatellites are non-essential satellite-like components associated with geminiviruses. The precise selective advantage to a geminivirus infection of an alphasatellite remains unclear. The ability of the cotton leaf curl Multan alphasatellite (CLCuMuA)-encoded replication-associated protein (Rep) to suppress TGS was investigated by using Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant Potato virus X (PVX) vector carrying CLCuMuA Rep resulted in restoration of GFP expression. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. Inoculation with PVX vectors harbouring a further six Rep proteins, encoded by genetically distinct alphasatellites, were similarly shown to result in 16-TGS plants with restored GFP expression. These results indicate that the alphasatellite-encoded Rep can restore the expression of a transcriptionally silenced GFP transgene in N. benthamiana, indicating that alphasatellites are involved in overcoming host defence.

1 Microinjection of CPE-Binding Protein Polyadenylated mRNA Increases Developmental Competence of Bovine Oocytes In Vitro

Developmental competence is acquired during oocyte growth and maturation while oocytes undergo both nuclear and cytoplasmic changes. Completion of oocyte maturation and subsequent embryo development relies mostly on maternally synthesised and stored mRNAs at the transcriptionally quiescent phase. The temporal and spatial post-transcriptional and translational regulation of the stored mRNA in mammalian oocyte cytoplasm is essential for developmental competence of oocytes and is often controlled via cytoplasmic polyadenylation. Cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) is required for polyadenylation of most mRNAs during oocyte maturation. It has been reported that in vitro-matured oocytes with high developmental competence showed an increased level of CPEB mRNA in oocyte cytoplasm. Thus, we hypothesise that the introduction of exogenous CPEB mRNA into in vitro-matured oocytes could increase their developmental capability. In this study, we first synthesised polyadenylated CPEB mRNAs by in vitro transcription. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and subjected to in vitro maturation for 21 h. After the removal of cumulus cells, matured oocytes were parthenogenetically activated (5 min in 5 mM ionomycin followed by 4 h in 2 mM DMAP with 5 mg mL−1 cycloheximide). Each activated oocyte was injected with 5 to 10 pL of poly(A)-RNA solution (400 ng μL−1; CPEB mRNA and green fluorescent protein (GFP) mRNA for the injection group or GFP mRNA for the control group) using a micromanipulator. After injection, the oocytes were cultured in SOF medium supplemented with amino acids for 8 days. No difference was observed in cleavage rate between CPEB and control group. However, the blastocyst rate was significantly higher in the CPEB group than in the control (24.9 ± 2.9% v. 15.0 ± 4.5%; P &lt; 0.05). Cleavage and blastocyst rates were analysed by one-way ANOVA. We also compared the gene expression profile of blastocysts derived from both groups. The blastocysts were collected individually and analysed by single-embryo RT-PCR. Twenty-two genes were selected for analysis based on their roles in genomic reprogramming and embryonic development and fell into 6 functional categories: growth regulatory factors, cell cycle regulation, imprinting, apoptosis, pluripotency and DNA methyltransferase. The single-embryo RT-PCR was performed using the Flex-Six integrated fluidic chip (Fluidigm Corp., South San Francsisco, CA, USA) on the BioMark platform (Fluidigm Corp.). Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. We found that 6 genes (H19, GRB10, DNMT1, CCNB1, CDK2, and SOX2) were up-regulated and 3 were down-regulated (DNMT2, BAX, and P53), along with the overexpression of CPEB gene (P &lt; 0.05). Our results demonstrate that developmental competence can be improved by injecting exogenous CPEB mRNA into in vitro-matured metaphase II cattle oocytes, which reaffirms the essential role of CPEB in early embryonic development.

Intra-luminal gene therapy in the porcine artery using a recombinant adeno-associated virus 9

The ability to improve or restore blood flow and promote healing in ischemic tissue has many potential clinical applications. Augmentation by direct delivery of growth factors may further enhance results, but requires a method for sustained delivery. In this study, we have tested the ability of adeno-associated virus 9 (AAV9) delivered within the lumen of a porcine artery to transfect the vessel and produce a desired product. The marker chosen was green fluorescent protein (GFP) (Ke et al., 2011).In 4 farm pigs the cranial tibial artery was surgically exposed. The vessel was temporarily clamped proximally, and divided distally. A cannula was placed intraluminally, and the arterial segment was injected with 1×10E13 particles of AAV9.CB7.CI.GFP·WPRE.rBG. At 14days the transfected cranial tibial artery as well as the liver, spleen and kidneys were harvested. ELISA and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were used to analyze the artery for GFP production.Significant GFP expression was seen in all transfected cranial tibial vessels, as determined by both GFP protein production (ELISA) and mRNA (RT-qPCR). No GFP was identified in liver, spleen or kidney, nor in the no-GFP control animal artery.Adeno-associated virus 9 is an appropriate vector for gene therapy experiments in the porcine artery model. This vector, and the intraluminal deliver method described result in robust gene expression at 2weeks without evident systemic spill of the virus. The ability to limit delivery of the gene to an isolated segment of vessel is desirable for future research applications.

Synthesis of an artificial Vitis vinifera miRNA 319e using overlapping long primers and its application for gene silencing

The conserved mechanism of action of micro-RNAs (miRNAs) as regulators of gene expression has allowed the use of artificial miRNAs (amiRNAs) as a powerful tool for candidate gene evaluation in plants. Based on the use of a Vitis vinifera miRNA molecule (i.e., vvi-miR319e), the present work presents a new methodology for designing artificial miR319e precursors (pre-amiR319e). As a proof of concept, we silenced the green fluorescent protein (GFP) gene in transgenic Nicotiana benthamiana plants. This methodology includes a two-step PCR reaction in which overlapping long primers allow for the complete generation of pre-amiR319e-GFP molecules that are adequate for recombination into Gateway vectors with no further requirements. The seed region in amiRNA was directed against the 3′-end portion of the GFP gene. Three groups of transformed N. benthamiana plants were generated: GFP-, amiR319e-GFP-, and GFP plus miR319e-GFP-expressing vectors. A similar group of wild-type plants was included. Confocal microscopy evaluation of these groups revealed strong silencing of the GFP phenotype in the double GFP plus amiR319e-GFP group. The molecular characterization of silenced plants was achieved via modified 5′RACE of the GFP mRNA and revealed the occurrence of a partial, 3′-end GFP mRNA molecule that was generated in planta. In addition, large-scale small RNA sequencing confirmed the occurrence of the expected 21-nt miR319e-GFP species and other 22- and 24-nt species that exhibited sequence relationships with the expected amiRNA. These results highlight the possibility of using vvi-MIR319 as a template for the generation of single amiRNAs as a tool for gene silencing in plants.

Intracellular delivery of messenger RNA by recombinant PP7 virus-like particles carrying low molecular weight protamine.

BackgroundCell-penetrating peptides (CPPs) have been widely used as carriers to transport different molecules into living cells, whereas messenger RNAs (mRNAs) have been utilized as target molecules for the prevention and treatment of various diseases. However, the instability of CPPs and mRNAs has limited their application. Bacteriophage PP7 virus-like particles (VLPs) may protect peptides and RNAs from degradation through displaying foreign peptides on their surface and encapsidating RNA linked with the pac site.ResultsIn this study, the cDNA of the PP7 coat protein single-chain dimer carrying low molecular weight protamine (LMWP) and the cDNA of green fluorescent protein (GFP) were inserted into two multiple cloning sites of pETDuet-1, respectively. PP7 VLPs carrying the LMWP peptide and GFP mRNA were subsequently expressed in Escherichia coli BL21 (DE3) with high yield and thermal stability, and were easily purified. The VLPs were also non-replicative, non-infectious, and non-toxic. Moreover, they penetrated the mouse prostate cancer cells RM-1 after 24 h incubation. Last, PP7 VLPs carrying the LMWP could encapsidate the GFP mRNA, which was translated into mature protein in mammalian cells.ConclusionsRecombinant PP7 VLPs can be used simultaneously as a targeted delivery vector for both peptides and mRNA due to their abilities to package RNA and display peptides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0274-9) contains supplementary material, which is available to authorized users.

Multilayered Nanoparticles for Gene Delivery Used to Reprogram Human Foreskin Fibroblasts to Neurospheres.

Polycationic nanocomplexes are a robust means for achieving nucleic acid condensation and efficient intracellular gene deliveries. To enhance delivery, a multilayered nanoparticle consisting of a core of electrostatically bound elements was used. These included a histone-mimetic peptides, poly-l-arginine and poly-d-glutamic acid was coated with silicate before surface functionalization with poly-l-arginine. Transfection efficiencies and duration of expression were similar when using green fluorescent protein (GFP) plasmid DNA (pDNA) or GFP mRNA. These nanoparticles demonstrated significantly higher (>100%) and significantly longer (15 vs. 4 days) transfection efficiencies in comparison to a commercial transfection agent (Lipofectamine 2000). Reprogramming of human foreskin fibroblasts using mRNA to the Sox2 transcription factor resulted in three-fold higher neurosphere formation in comparison to the commercial reagent. These results demonstrate the potential of these nanoparticles as ideal vectors for gene delivery.

Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression

Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics technology. The rescued recombinant viruses retained lentogenic pathotype and displayed delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer to the 3' end and gradually decreased as the gene was inserted closer to the 5' end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the non-coding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.

The β-actin gene promoter of rohu carp (Labeo rohita) drives reporter gene expressions in transgenic rohu and various cell lines, including spermatogonial stem cells.

We previously characterized the β-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The β-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of β-actin gene expression across eukaryotes. Furthermore, the isolated β-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.