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Background: Non-conventional yeasts hold significant potential as biorefinery cell factories for microbial bioproduction. Currently, gene editing systems used for these yeasts rely on antibiotic and auxotrophic selection mechanisms. However, the drawbacks of antibiotics, including high costs, environmental concerns, and the dissemination of resistance genes, make them unsuitable for large-scale industrial fermentation. For auxotrophic selection system, the engineered strains harboring auxotrophic marker genes are typically supplemented with complex nutrient-rich components instead of precisely defined synthetic media in large-scale industrial fermentations, thus lack selection pressure to ensure the stability of heterologous metabolic pathways. Therefore, it is a critical to explore alternative selection systems that can be adapted for large-scale industrial fermentation. Results: Here, a novel glucose-dependent selection system was developed in a high pullulan-producing non-conventional strain A. melanogenum P16. The system comprised a glucose-deficient chassis cell Δpfk obtained through the knockout of the phosphofructokinase gene (PFK) and a series of chromosomal integration plasmids carrying a selection marker PFK controlled by different strength promoters. Utilizing the green fluorescent protein gene (GFP) as a reporter gene, this system achieved a 100% positive rate of transformation, and the chromosomal integration numbers of GFP showed an inverse relationship with promoter strength, with a customizable copy number ranging from 2 to 54. More importantly, the chromosomal integration numbers of target genes remained stable during successive inoculation and fermentation process, facilitated simply by using glucose as a cost-effective and environmental-friendly selectable molecule to maintain a constant and rigorous screening pressure. Moreover, this glucose-dependent selection system exhibited no significant effect on cell growth and product synthesis, and the glucose-deficient related selectable marker PFK has universal application potential in non-conventional yeasts. Conclusion: Here, we have developed a novel glucose-dependent selection system to achieve customizable and stable multilocus chromosomal integration of target genes. Therefore, this study presents a promising new tool for genetic manipulation and strain enhancement in non-conventional yeasts, particularly tailored for industrial fermentation applications. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime. Methods: In this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking. Results: Firstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively. Conclusions: Taken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC. [ABSTRACT FROM AUTHOR]

Summary: Inducible expression systems can overcome the trade‐off between high‐level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature‐inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature‐sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light‐dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature‐dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pR promoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat‐inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost‐effective and does not depend on chemical inducers. [ABSTRACT FROM AUTHOR]

The bottleneck in drug discovery for central nervous system diseases is the absence of effective systemic drug delivery technology for delivering therapeutic drugs into the brain. Despite the advances in the technology used in drug discovery, such as Adeno-Associated Virus (AAV) vectors, the development of drugs for central nervous system diseases remains challenging. In this manuscript, we describe, for the first time, the development of a workflow to generate a novel brain-targeted drug delivery system that involves the generation of genetically engineered exosomes by first selecting various functional AAV capsid-specific peptides (collectively called CAPs) known to be involved in brain-targeted high-expression gene delivery, and then expressing the CAPs in frame with lysosome-associated membrane glycoprotein (Lamp2b) followed by expressing CAP-Lamp2b fusion protein on the surface of mesenchymal stem cell-derived exosomes, thus generating CAP-exosomes. Intravenous injection of green fluorescent protein (GFP) gene-loaded CAP-exosomes in mice transferred the GFP gene throughout the CNS as measured by monitoring brain sections for GFP expression with confocal microscopy. GFP gene transfer efficiency was at least 20-fold greater than that of the control Lamp2b-exosomes, and GFP gene transduction to mouse liver was low. [ABSTRACT FROM AUTHOR]

Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) is a powerful gene therapy vector that has been used in several FDA-approved therapies as well as in multiple clinical trials. This vector has high therapeutic versatility with the ability to deliver genetic payloads to a variety of human tissue types, yet there is currently a lack of transgene expression control once the virus is administered. There are also times when transgene expression is too low for the desired therapeutic outcome, necessitating high viral dose administration resulting in possible immunological complications. Herein, we validate a chemically controllable AAV transgene expression technology in vitro that utilizes bifunctional molecules known as chemical epigenetic modifiers (CEMs). These compounds employ endogenous epigenetic machinery to specifically enhance transgene expression of episomal DNA. A recombinant AAV (rAAV) was designed to both deliver the reporter transgene as well as deliver a synthetic zinc finger (ZFs) protein fused to FK506 binding protein (FKBP). These synthetic ZFs target a DNA-binding array sequence upstream of the promoter expressing the AAV transgene to specifically enhance AAV transgene expression in the presence of a CEM. The transcriptional activating compound CEM87 functions by recruiting the epigenetic transcription activator bromodomain-containing protein 4 (BRD4), increasing AAV transgene activity up to fivefold in a dose-dependent manner in HEK293T cells. The highest levels of transgene product activity are seen 24 h following CEM87 treatment. Additionally, the CEM87-mediated enhancement of different transgene products with either Luciferase or green fluorescent protein (GFP) was observed in multiple cell lines and enhancement of transgene expression was capsid serotype independent. The impact of CEM87 activity can be disrupted through drug removal or chemical recruitment site competition with FK506, thus demonstrating the reversibility of the impact of CEM87 on transgene expression. Collectively, this chemically controllable rAAV transgene technology provides temporal gene expression control that could increase the safety and efficiency of AAV-based research and therapies. [ABSTRACT FROM AUTHOR]

Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light. [ABSTRACT FROM AUTHOR]

Much of our understanding of the intracellular regulation of macroautophagy/autophagy comes from in vitro studies. However, there remains a paucity of knowledge about how this process is regulated within different tissues during development, aging and disease in vivo. Because upregulation of autophagy is considered a promising therapeutic strategy for the treatment of diverse disorders, it is vital that we understand how this pathway functions in different tissues and this is best done by in vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate how tissue-specific changes in flux and cell-autonomous versus non-cell-autonomous effects alter disease progression. To this end, we have developed an inducible expression system to up- or downregulate autophagy in vivo, in zebrafish. We have used a modified version of the Gal4-UAS expression system to allow inducible expression of autophagy up- or downregulating transgenes by addition of tamoxifen. Using this inducible expression system, we have tested which transgenes robustly up- or downregulate autophagy and have validated these tools using Lc3-II blots and puncta analysis and disease rescue in a zebrafish model of neurodegeneration. These tools allow the temporal control of autophagy via the administration of tamoxifen and spatial control via tissue or cell-specific ERT2-Gal4 driver lines and will enable the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation and neurodegeneration in vivo. Abbreviations: ANOVA: analysis of variance; Atg: autophagy related; Bcl2l11/Bim: BCL2 like 11; d.p.f.: days post-fertilization; Cryaa: crystallin, alpha a: DMSO: dimethyl sulfoxide; Elavl3: ELAV like neuron-specific RNA binding protein 3; ER: estrogen receptor; ERT2: modified ligand-binding domain of human ESR1/estrogen receptor α; Gal4: galactose-responsive transcription factor 4; GFP: green fluorescent protein; h.p.f.: hours post-fertilization; HSP: heat-shock protein; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; SD: standard deviation; SEM: standard error of the mean; UAS: upstream activating sequence; Ubb: ubiquitin b [ABSTRACT FROM AUTHOR]

CRISPR/Cas9‐mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock‐in (KI) efficiency is critical for fully implementing TI‐mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site‐independent transgene expression. UCOE KI cassettes were randomly integrated into CHO‐K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO‐K1 random pool harboring 5′2.2A2UCOE‐CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5′2.2A2UCOE‐CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology‐independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high‐production targeted integrants by mitigating the selection of genomic hot spots. [ABSTRACT FROM AUTHOR]

Broad host range (BHR) expression vector is a vital tool in molecular biology research and application. Currently, most of the plasmid vectors used in Agrobacterium spp. are binary vectors that are designed for plant transformation, and very few are designed for expressing transgenes in Agrobacterium spp. Class 1 integrons are common genetic elements that allow for the efficient capture and expression of antibiotic resistance genes, especially in Gram-negative bacteria. One of its compound promoters, PcS + P2, was used in this study and has been reported to be the strongest class 1 integron constitutive promoter; it is referred to as "integron promoter" (Pint) henceforth. Herein, we created two versions of isopropyl–d-thiogalactopyranoside (IPTG)-inducible promoters by substituting and/or inserting lacO sequence(s) into Pint. These inducible promoters, which possess different degrees of stringency and inducibility, were used to construct two broad host range expression vectors (pWK102 and pWK103) based on the versatile pGREEN system. This allows them to be stably maintained and replicated in both Escherichia coli and Agrobacterium tumefaciens. Functional validation of these vectors was performed by the expression of the reporter gene, superfolder green fluorescent protein (sfGFP), which was cloned downstream of these promoters. Due to the strong induction and tunable expression of a transgene located downstream to the inducible integron promoter, these vectors may be useful for heterologous gene expression in both E. coli and A. tumefaciens, thus facilitating recombinant protein production and genetic studies in Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. Therefore, achieving tissue-specific expression of transgenes in the CA would be beneficial for mosquito research and control. Different CA promoters have been used to drive transgene expression in Drosophila, but mosquito CA-specific promoters have not been identified. Using the CRISPR/Cas9 system, we integrated transgenes encoding the reporter green fluorescent protein (GFP) close to the transcription start site of juvenile hormone acid methyl transferase (JHAMT), a locus encoding a JH biosynthetic enzyme, specifically and highly expressed in the CA of Aedes aegypti mosquitoes. Transgenic individuals showed specific GFP expression in the CA but failed to reproduce the full pattern of jhamt spatiotemporal expression. In addition, we created GeneSwitch driver and responder mosquito lines expressing an inducible fluorescent marker, enabling the temporal regulation of the transgene via the presence or absence of an inducer drug. The use of the GeneSwitch system has not previously been reported in mosquitoes and provides a new inducible binary system that can control transgene expression in Aedes aegypti. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells (MSCs) driven gene directed enzyme prodrug therapy is a promising approach to deliver therapeutic agents to target heterogenous solid tumours. To democratize such a therapy, cryopreservation along with cold chain transportation is an essential part of the logistical process and supply chain. Previously, we have successfully engineered MSCs by a non-viral DNA transfection approach for prolonged and exceptionally high expression of the fused transgene cytosine deaminase, uracil phosphoribosyl transferase and green fluorescent protein (CD::UPRT::GFP). The aim of this study was to determine the effects of cryopreservation of MSCs engineered to highly overexpress this cytoplasmic therapeutic transgene. Methods: Modified MSCs were preserved in a commercially available, GMP-grade cryopreservative—CryoStor10 (CS10) for up to 11 months. Performance of frozen-modified MSCs was compared to freshly modified equivalents in vitro. Cancer killing potency was evaluated using four different cancer cell lines. Migratory potential was assessed using matrigel invasion assay and flow cytometric analysis for CXCR4 expression. Frozen-modified MSC was used to treat canine patients via intra-tumoral injections, or by intravenous infusion followed by a daily dose of 5-flucytosine (5FC). Results: We found that cryopreservation did not affect the transgene expression, cell viability, adhesion, phenotypic profile, and migration of gene modified canine adipose tissue derived MSCs. In the presence of 5FC, the thawed and freshly modified MSCs showed comparable cytotoxicity towards one canine and three human cancer cell lines in vitro. These cryopreserved cells were stored for about a year and then used to treat no-option-left canine patients with two different types of cancers and notably, the patients showed progression-free interval of more than 20 months, evidence of the effectiveness in treating spontaneously occurring cancers. Conclusion: This study supports the use of cryopreserved, off-the-shelf transiently transfected MSCs for cancer treatment. [ABSTRACT FROM AUTHOR]

Gene electrotransfer (GET) is a widely used method for nucleic acids' delivery into cells. We explored, evaluated, and demonstrated the potential use of different pulse durations for introducing plasmid DNA (pDNA) into cells in vitro and compared the efficiency and dynamics of transgene expression after GET. We performed experiments on cell suspensions of 1306 fibroblasts and C2C12 myoblasts with four ranges of pulse durations (nanosecond, high frequency bipolar (HF-BP), and micro- and millisecond). Six different concentrations of pDNA encoding green fluorescent protein were used. We show that GET can be achieved with nanosecond pulses with a low pulse repetition rate (10 Hz). The GET's efficiency depends on the pDNA concentration and cell line. Time dynamics of transgene expression are comparable between millisecond, microsecond, HF-BP, and nanosecond pulses but depend greatly on cell line. Lastly, based on the data obtained in the experiments of pDNA concentration effect on GET the model of the probability of pDNA and cell membrane contact during GET was developed. The model shows that pDNA migration is dominated by diffusion for nanosecond and HF-BP pulses and by electrophoresis for micro- and millisecond pulses. Modeling results can provide valuable guidance for further experiments and interpretations of the results obtained by various pulse protocols. [ABSTRACT FROM AUTHOR]

Transgenesis with the meganuclease I-SceI is a safe and efficient method, but the underlying mechanisms remain unclear due to the lack of information on transgene localization. Using I-SceI, we previously developed a transgenic Xenopus tropicalis line expressing enhanced green fluorescent protein driven by the neural crest-specific snai2 promoter/enhancer, which is a powerful tool for studying neural crest development and craniofacial morphogenesis. Here, we carried out whole-genome shotgun sequencing for the snai2:eGFP embryos to identify the transgene integration sites. With a 19x sequencing coverage, we estimated that 6 copies of the transgene were inserted into the Xenopus tropicalis genome in the hemizygous transgenic embryos. Two transgene integration loci adjacent to each other were identified in a noncoding region on chromosome 1, possibly as a result of duplication after a single transgene insertion. Interestingly, genomic DNA at the boundaries of the transgene integration loci contains short sequences homologous to the I-SceI recognition site, suggesting that the integration was not random but probably mediated by sequence homology. To our knowledge, our work represents the first genome-wide sequencing study on a transgenic organism generated with I-SceI, which is useful for evaluating the potential genetic effects of I-SceI-mediated transgenesis and further understanding the mechanisms underlying this transgenic method. [ABSTRACT FROM AUTHOR]

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for recombinant therapeutic protein (RTP) production. Currently, the RTP yields need to increase further to meet the market needs and reduce costs. In this study, three stabilizing and anti-repressor (SAR) elements from the human genome were selected, including human SAR7, SAR40, and SAR44 elements. SAR elements were cloned upstream of the promoter in the eukaryotic vector, followed by transfection into CHO cells, and were screened under G418 pressure. Flow cytometry was used to detect enhanced green fluorescent protein (eGFP) expression levels. The gene copy numbers and mRNA expression levels were determined through quantitative real-time PCR. Furthermore, the effect of the stronger SAR elements on adalimumab was investigated. The results showed that transgene expression levels in the SAR-containing vectors were higher than that of the control vector, and SAR7 and SAR40 significantly increased and maintained the long-term expression of the transgene in CHO cells. In addition, the transgene expression level increase was related with gene copy numbers and mRNA expression levels. Collectively, SAR elements can enhance the transgene expression and maintain the long-term expression of a transgene in transfected CHO cells, which may be used to increase recombinant protein production in CHO cells.

Virus-like particles (VLP) are a promising tool for intracellular gene delivery, yet their potential in ocular gene therapy remains underexplored. In this study, we bridged this knowledge gap by demonstrating the successful generation and application of vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped mouse PEG10 (MmPEG10)-VLP for intraocular mRNA delivery. Our findings revealed that PEG10-VLP can efficiently deliver GFP mRNA to adult retinal pigment epithelial cell line-19 (ARPE-19) cells, leading to transient expression. Moreover, we showed that MmPEG10-VLP can transfer SMAD7 to inhibit epithelial-mesenchymal transition (EMT) in RPE cells effectively. In vivo experiments further substantiated the potential of these vectors, as subretinal delivery into adult mice resulted in efficient transduction of retinal pigment epithelial (RPE) cells and GFP reporter gene expression without significant immune response. However, intravitreal injection did not yield efficient ocular expression. We also evaluated the transduction characteristics of MmPEG10-VLP following intracameral delivery, revealing transient GFP protein expression in corneal endothelial cells without significant immunotoxicities. In summary, our study established that VSVG pseudotyped MmPEG10-based VLP can transduce mitotically inactive RPE cells and corneal endothelial cells in vivo without triggering an inflammatory response, underscoring their potential utility in ocular gene therapy. • MmPEG10-VLP can efficiently deliver mRNA to ARPE-19 cells, leading to transient expression. • Subretinal delivery of MmPEG10-VLP resulted in efficient transduction of RPE cells without significant immune response. • Intracameral delivery of MmPEG10-VLP revealed transgene expression in corneal endothelial cells. [ABSTRACT FROM AUTHOR]

The phenomenon of RNA interference (RNAi) is widely used to develop new approaches for crop improvement and plant protection. Recent investigations show that it is possible to downregulate plant transgenes, as more prone sequences to silencing than endogenous genes, by exogenous application of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs). However, there are scarce data on the specificity of exogenous RNAs. In this study, we explored whether plant transgene suppression is sequence-specific to exogenous dsRNAs and whether similar effects can be caused by exogenous DNAs that are known to be perceived by plants and induce certain epigenetic and biochemical changes. We treated transgenic plants of Arabidopsis thaliana bearing the neomycin phosphotransferase II (NPTII) transgene with specific synthetic NPTII-dsRNAs and non-specific dsRNAs, encoding enhanced green fluorescent protein (EGFP), as well as with DNA molecules mimicking the applied RNAs. None of the EGFP-dsRNA doses resulted in a significant decrease in NPTII transgene expression in the NPTII-transgenic plants, while the specific NPTII-dsRNA significantly reduced NPTII expression in a dose-dependent manner. Long DNAs mimicking dsRNAs and short DNA oligonucleotides mimicking siRNAs did not exhibit a significant effect on NPTII transgene expression. Thus, exogenous NPTII-dsRNAs induced a sequence-specific and RNA-specific transgene-suppressing effect, supporting external application of dsRNAs as a promising strategy for plant gene regulation. [ABSTRACT FROM AUTHOR]

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. [ABSTRACT FROM AUTHOR]

Conventional cancer therapies, including surgery, radiotherapy, and chemotherapy, are not tumor site-specific and have cytotoxic and harmful side effects for normal cells. Mesenchymal stem cells (MSCs), due to their tumor-tropism migration property, are a promising alternative to deliver and produce antitumor agents. However, MSCs are difficult-to-transfect cells, and introducing the exogenous therapeutic gene into MSCs is challenging yet needs improvement. Transfection using chemical reagents, including Lipofectamine, is more convenient and less cytotoxic compared with different methods of introducing exogenous DNA into MSCs. Nonetheless, the major limitation of Lipofectamine is low transfection efficiency in MSCs. Therefore, the purpose of this study was to evaluate and suggest the optimum quantities of lipoplex components to enhance the transfection efficiency of human adipose tissue-derived MSCs (hASCs). Finding the best transgene expression time point and the optimum concentration of G-418 for antibiotic-based selection was another goal of this study. hASCs were transfected in a series of experiments with altering the quantities of Lipofectamine LTX® (LipLTX), the related “PLUS” reagent, and a plasmid DNA (pDNA) expressing the enhanced green fluorescent protein (eGFP). After transfection, the percentage of eGFP-expressing cells was evaluated using fluorescence microscopy and ImageJ software in 12-hour intervals for 48 hours. Also, the viability of hASCs exposed to different concentrations of G-418 was measured using an MTT assay. The results demonstrated that a combination of 2 µL Lip-LTX, 0.75 µL of its “PLUS” reagent, and 0.75 g pDNA (6484 bp) improve the transfection efficiency of hASCs (23.75%), and the best period for evaluation of fluorescence for these cells is 12 to 24h post-transfection. Also, the optimum concentration of G-418 for antibiotic-based selection of hASCs was 0.25mg/mL. In conclusion, this study indicates that the setting up of optimized quantities of lipoplex components and the golden time of evaluation for transgene expression could increase the possibility of transgene expression in hASCs before beginning research and clinical application. Also, the definition of optimal dose of selection antibiotic for purification of transfected hASCs seems to be necessary for maximum transgene expression effects in the cell population. [ABSTRACT FROM AUTHOR]

The Tet‐ON system is an important molecular tool for temporally and spatially‐controlled inducible gene expression. Here, we developed a Tet‐ON system to induce transgene expression specifically in the rod photoreceptors of medaka fish. Our modified reverse tetracycline‐controlled transcriptional transactivator (rtTAm) with 5 amino acid substitutions dramatically improved the leakiness of the transgene in medaka fish. We generated a transgenic line carrying a self‐reporting vector with the rtTAm gene driven by the Xenopus rhodopsin promoter and a tetracycline response element (TRE) followed by the green fluorescent protein (GFP) gene. We demonstrated that GFP fluorescence was restricted to the rod photoreceptors in the presence of doxycycline in larval fish (9 days post‐fertilization). The GFP fluorescence intensity was enhanced with longer durations of doxycycline treatment up to 72 h and in a dose‐dependent manner (5–45 μg/ml). These findings demonstrate that the Tet‐ON system using rtTAm allows for spatiotemporal control of transgene expression, at least in the rod photoreceptors, in medaka fish. [ABSTRACT FROM AUTHOR]

Bromodomain (BRD) proteins are histone code interpreters that recognize acetylated lysines and link the dynamic state of chromatin with the transcriptional machinery. Here, we demonstrate that ablation of the Brd4 gene in primary mouse bone marrow–derived mesenchymal stem cells via a conditional Brd4fl/fl allele suppresses osteogenic lineage commitment. Remarkably, loss of Brd4 function also enhances expression of genes in engineered adenoviral vectors, including Cre recombinase and green fluorescent protein (GFP). Similarly, vector‐based expression of BMP2 mRNA and protein levels are enhanced upon Brd4 depletion in cells transduced with an adenoviral vector that expresses BMP2 (Ad‐BMP2). Importantly, Brd4 depletion in MC3T3‐E1 and human adipose‐derived mesenchymal stem cells (AMSCs) transduced with Ad‐BMP2 enhances osteogenic differentiation of naïve MC3T3‐E1 cells via paracrine mechanisms based on transwell and conditioned medium studies. Our studies indicate that Brd4 depletion enhances adenoviral transgene expression in mammalian cells, which can be leveraged as a therapeutic strategy to improve viral vector‐based gene therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

The suprachoroid is a potential space located between the sclera and choroid of the eye, which provides a novel route for ocular drug or viral vector delivery. Suprachoroidal injection of adeno-associated virus (AAV)8 using transscleral microneedles enables widespread transgene expression in eyes of nonhuman primates, but may cause intraocular inflammation. We characterized the host humoral and cellular immune responses after suprachoroidal delivery of AAV8 expressing green fluorescent protein (GFP) in rhesus macaques, and found that it can induce mild chorioretinitis that resolves after systemic corticosteroid administration, with recovery of photoreceptor morphology, but persistent immune cell infiltration after 3 months, corresponding to a loss of GFP expression from retinal pigment epithelial cells, but persistent expression in scleral fibroblasts. Suprachoroidal AAV8 triggered B cell and T cell responses against GFP, but only mild antibody responses to the viral capsid compared to intravitreal injections of the same vector and dose. Systemic biodistribution studies showed lower AAV8 levels in liver and spleen after suprachoroidal injection compared with intravitreal delivery. Our findings suggest that suprachoroidal AAV8 primarily triggers host immune responses to GFP, likely due to sustained transgene expression in scleral fibroblasts outside the blood–retinal barrier, but elicits less humoral immune reactivity to the viral capsid than intravitreal delivery due to lower egress into systemic circulation. As GFP is not native to primates and not a clinically relevant transgene, suprachoroidal AAV delivery of human transgenes may have significant translational potential for retinal gene therapy. [ABSTRACT FROM AUTHOR]

Viral vectors made from adeno-associated virus (AAV) have emerged as preferred tools in basic and translational neuroscience research to introduce or modify genetic material in cells of interest. The use of viral vectors is particularly attractive in nontransgenic species, such as nonhuman primates. Injection of AAV solutions into the cerebrospinal fluid is an effective method to achieve a broad distribution of a transgene in the central nervous system. In this study, we conducted injections of AAV9-PHP.B, a recently described AAV capsid mutant, in the lateral ventricle of mice and rhesus macaques. To enhance the expression of the transgene (the tag protein emerald green fluorescent protein [EmGFP]), we used a gene promoter that confers high neuron-specific expression of the transgene, the human synapsin 1 (SYN1) promoter. The efficacy of the viral vector was first tested in mice. Our results show that intracerebroventricular injections of AAV9-PHP.B SYN1-EmGFP-woodchuck hepatitis virus posttranscriptional regulatory element resulted in neuronal EmGFP expression throughout the mice and monkey brains. We have provided a thorough characterization of the brain regions expressing EmGFP in both species. EmGFP was observed in neuronal cell bodies over the whole cerebral cortex and in the cerebellum, as well as in some subcortical regions, including the striatum and hippocampus. We also observed densely labeled neuropil in areas known to receive projections from these regions. Double fluorescence studies demonstrated that EmGFP was expressed by several types of neurons throughout the mouse and monkey brain. Our results demonstrate that a single injection in the lateral ventricle is an efficient method to obtain transgene expression in many cortical and subcortical regions, obviating the need of multiple intraparenchymal injections to cover large brain areas. The use of intraventricular injections of AAV9-PHP.B SYN1-EmGFP could provide a powerful approach to transduce widespread areas of the brain and may contribute to further development of methods to genetically target-specific populations of neurons. [ABSTRACT FROM AUTHOR]

Nonpathogenic retroviruses of the Spumaretrovirinae subfamily can persist long term in the cytoplasm of infected cells, completing their life cycle only after the nuclear membrane dissolves at the time of cell division. Since the targeting of slowly dividing cancer cells remains an unmet need in oncolytic virotherapy, we constructed a replication-competent foamy virus vector (oFV) from the genomes of two chimpanzee simian foamy viruses (PAN1 and PAN2) and inserted a green fluorescent protein (GFP) transgene in place of the bel-2 open reading frame. oFV-GFP infected and propagated with slow kinetics in multiple human tumor cell lines, inducing a syncytial cytopathic effect. Infection of growth-arrested MRC5 cells was not productive, but oFV genomes persisted in the cytoplasm, and the productive viral life cycle resumed when cell division was later restored. In vivo, the virus propagated extensively in intraperitoneal ovarian cancer xenografts, slowing tumor growth, significantly prolonging survival of the treated mice, and sustaining GFP transgene expression for at least 45 days. Our data indicate that oFV is a promising new replication-competent viral and gene delivery platform for efficient targeting of the most fundamental trait of cancer cells, their ability to sustain chronic proliferation. [ABSTRACT FROM AUTHOR]

Summary: Reverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields. Genetically encoded fluorescent proteins (FPs) have revolutionized plant biology paradigms in gene expression, protein trafficking and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 25 years ago, modern phenomics has largely ignored fluorescence as a transgene expression device despite the burgeoning FP colour palette available to plant biologists. Here, we show a new platform for stand‐off imaging of plant canopies expressing a wide variety of FP genes. The platform—the fluorescence‐inducing laser projector (FILP)—uses an ultra‐low‐noise camera to image a scene illuminated by compact diode lasers of various colours, coupled with emission filters to resolve individual FPs, to phenotype transgenic plants expressing FP genes. Each of the 20 FPs screened in plants were imaged at >3 m using FILP in a laboratory‐based laser range. We also show that pairs of co‐expressed fluorescence proteins can be imaged in canopies. The FILP system enabled a rapid synthetic promoter screen: starting from 2000 synthetic promoters transfected into protoplasts to FILP‐imaged agroinfiltrated Nicotiana benthamiana plants in a matter of weeks, which was useful to characterize a water stress‐inducible synthetic promoter. FILP canopy imaging was also accomplished for stably transformed GFP potato and in a split‐GFP assay, which illustrates the flexibility of the instrument for analysing fluorescence signals in plant canopies. [ABSTRACT FROM AUTHOR]

Wheat (Triticum aestivum L.) is a major food crop worldwide whose complex and very large genome hinders stable genetic transformation. An optimized method for transformation by particle bombardment is therefore greatly needed, both in general and for Korean commercial wheat varieties, which have not yet been successfully transformed. In this study, we describe the stable transformation of the Korean commercial wheat variety Keumkang and the DH20, which lacks the Glu-B3 and Gli-B1 loci (both related to gluten production), via particle bombardment of a vector harboring the enhanced green fluorescent protein (eGFP) gene. We confirmed the insertion of the eGFP transgene into the genome of primary (T0) transformants by genomic PCR, RT-qPCR, and fluorescence imaging. Our estimated average transformation efficiencies were 4.4% for Keumkang and 3.5% for DH20 in the T0 generation. We also validated the stable expression of the transgene in the T1 generation. We hypothesize that donor plant health, centrifugation, and high-sucrose pre-treatment increased the transformation efficiency reported here. Taken together, these results describe a useful method for transforming Korean commercial wheat varieties, including Keumkang and DH20, that will allow the manipulation of gene expression via such techniques as RNA interference, overexpression, and genome editing. [ABSTRACT FROM AUTHOR]

Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand topically-induced, systemic transgene silencing in Nicotiana benthamiana. A previous report details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event. This investigation revealed an inadvertent co-integration of part of a bacterial transposase in this line. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous GFP16C lines produced for this study ranged from 50–72% of the homozygous GFP16C line. GFP expression was equivalent to GFP16C in a two-copy event. Local GFP silencing was observed in all transgenic and GFP16C hemizygous lines after topical application of carbon dot-based formulations containing a GFP targeting dsRNA. The GFP16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of topically-induced, systemic transgene silencing in N. benthamiana. [ABSTRACT FROM AUTHOR]

The article offers information on a study that explores the enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles. It mentions that systemic AAV (adeno-associated virus) gene therapy as a promising approach for the treatment of inborn errors of metabolism.

In the past, various species of the unicellular algal genus Micrasterias (Zygnematophyceae) have been used for research in the fields of cell biology and physiology. Relatively large cell size, highly differentiated cell shape and a remarkable evolutionary position make Micrasterias interesting, especially for cytomorphogenetic studies. However, within this genus a model organism enabling molecular research has not yet been established. Micrasterias radians var. evoluta (W.B.Turner) Krieger allows easy culturing under axenic conditions and performs its whole life cycle in vitro thus fulfilling two important prerequisites for a model organism. In this work resources allowing transient expression of transgenes were developed. First, axenic lines were obtained by the treatment of zygospores with a cocktail of antibiotics followed by germination and regeneration. In order to allow transgene expression we isolated the 5′ -flanking region of the M. radians var. evoluta Actin1 gene (MrACT1) and fused it to the green fluorescent protein (GFP). A higher promoter activity compared with various heterologous promoters regarding GFP expression was observed. Transient transgene expression was achieved by polyethylene glycol (PEG)-mediated protoplast transformation, yielding a rate of 3.9% transformed cells per surviving protoplasts. Transgene expression was also achieved by particle bombardment of vegetative cells. Employing the established protocol, a Lifeact-GFP fusion protein for labelling F-actin was expressed, allowing visualization of the actin cytoskeleton in M. radians var. evoluta. [ABSTRACT FROM AUTHOR]

• The generation of homogeneously low expressing recombinant cell populations is a technical challenge. • The generation of multiple cell populations expressing slightly different low levels of the gene of interest is an additional challenge. • A vector construct is described that allows to establish homogeneous cell populations with tunable levels of a recombinant gene of interest. • An example is provided that shows the killing of target cell populations with a BEAT antibody as function of different receptor expression levels. We describe here a vector construct to establish homogeneous cell populations expressing a recombinant gene of interest (GOI) at tuneable levels, including low expression levels that are difficult to generate using standard cell line development techniques. This is achieved using a tricistronic mRNA that contains an open reading frame for the gene of interest, a first internal ribosome entry site (IRES), an open reading frame for a fluorescent reporter protein (such as green fluorescent protein, GFP), a second IRES and an open reading for an antibiotic resistance gene (such as puromycin N-acetyl-transferase, PAC). The resistance gene allows convenient selection of stable cell populations. The fluorescent reporter protein allows convenient homogeneity and expression stability assessments of the cell line. The expression level of the GOI can be adjusted by using different start codons for the open reading frame. These alternate start codons will initiate the translation of the GOI with different efficiency, leading to cell populations expressing different levels of the GOI, and similar levels of the fluorescent reporter through the first IRES and the puromycin resistance gene through the second IRES to the GOI. Such cell populations are useful tools, for instance to assess the safety of potent targeted therapeutics, as they allow the simplified generations of homogenous cell populations with different levels of target protein expression between populations. [ABSTRACT FROM AUTHOR]

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human‐like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT‐PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate‐early enhancer combined with the chicken beta‐actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33 °C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta‐actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells. [ABSTRACT FROM AUTHOR]

Retrogradely‐transducing viral vectors are versatile tools for anatomical and functional interrogations of neural circuits. These vectors can be applied in nonhuman primates (NHPs), powerful model species for neuroscientific studies with limited genetic tractability, but limited data are available regarding the tropism and transgene expression patterns of such viruses after injections in NHP brains. Consequently, NHP researchers must often rely on related data available from other species for experimental planning. To evaluate the suitability of rAAV2‐retro in the NHP basal ganglia, we studied the transgene expression patterns at the light and electron microscope level after injections of rAAV2‐retro vector encoding the opsin Jaws conjugated to a green fluorescent protein (GFP) in the putamen of rhesus macaques. For inter‐species comparison, we injected the same vector in the rat dorsal striatum. In both species, GFP expression was observed in numerous cortical and subcortical regions with known striatal projections. However, important inter‐species differences in pathway transduction were seen, including labeling of the intralaminar thalamostriatal projection in rats, but not monkeys. Electron microscopic ultrastructural observations within the basal ganglia revealed GFP labeling in both postsynaptic dendrites and presynaptic axonal terminals; the latter likely derived from anterograde transgene transport in neurons that project to the striatum, and from collaterals of these neurons. Our results suggest that certain neural pathways may be refractory to transduction by retrograde vectors in a species‐specific manner, highlighting the need for caution when determining the suitability of a retrograde vector for NHP studies based solely on rodent data. [ABSTRACT FROM AUTHOR]

Background: With the goal of labeling and manipulating the zebrafish hypothalamus, we sought to target a green fluorescent protein (gfp) transgene to the expression domains of nkx2.4b, a gene expressed during hypothalamic and thyroid development. We combined transcription activator‐like effector nucleases (TALENs)‐mediated mutagenesis with a targeting construct to enable insertion of a gfp transgene into the endogenous nkx2.4b genomic locus. Results: Injection of TALENs targeted to the first exon of nkx2.4b created a predicted null allele, and homozygous mutant embryos displayed loss of thyroid markers. From embryos injected with both TALENs and a targeting construct carrying a gfp transgene, we recovered a line in which GFP was expressed specifically in the hypothalamus and thyroid. Fish homozygous for this allele lacked exon 1 of nkx2.4b and exhibited hypothyroid phenotypes. Conclusions: By combining TALENs injections with a targeting construct that contained a gfp transgene, we were able to recover an allele in which GFP is expressed in the nkx2.4b expression domains, with homozygous phenotypes suggesting the creation of a loss‐of‐function transgenic line. These results demonstrate the creation of a useful tool for studying hypothalamus and thyroid development. Key Findings: Transgene targeting to the nkx2.4b locus in zebrafish.Disruption of the nkx2.4b gene by TALENs mutagenesis and transgene insertion both result in hypothyroid phenotypes.The inserted transgene expresses GFP in the hypothalamus and thyroid. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean. [ABSTRACT FROM AUTHOR]

The striatum, the main input structure of the basal ganglia, is critical for action selection and adaptive motor control. To understand the neuronal mechanisms underlying these functions, an analysis of microcircuits that compose the striatum is necessary. Among these, cholinergic interneurons (ChIs) provide intrinsic striatal innervation whose dysfunction is implicated in neuropsychiatric diseases, such as Parkinson's disease and Tourette syndrome. The ability to experimentally manipulate the activity of ChIs is critical to gain insights into their contribution to the normal function of the striatum and the emergence of behavioral abnormalities in pathological states. In this study, we generated and tested CAV-pChAT-GFP, a replication-defective canine adenovirus type 2 (CAV-2) vector carrying the green fluorescent protein (GFP) sequence under the control of the human choline acetyltransferase (ChAT) promoter. We first tested the potential specificity of CAV-pChAT-GFP to label striatal ChIs in a rat before performing experiments on two macaque monkeys. In the vector-injected rat and monkey striatum, we found that GFP expression preferentially colocalized with ChAT-immunoreactivity throughout the striatum, including those from local circuit interneurons. CAV-2 vectors containing transgene driven by the ChAT promoter provide a powerful tool for investigating ChI contributions to circuit function and behavior in nonhuman primates. [ABSTRACT FROM AUTHOR]

Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3' untranslated region (3' UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy. [ABSTRACT FROM AUTHOR]

The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65–85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level. [ABSTRACT FROM AUTHOR]

Objectives: Previously, we have found that the matrix attachment region (MAR) may confer a 'distance effect' on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism. Results: Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers. Conclusions: A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system. [ABSTRACT FROM AUTHOR]

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 3ʹ 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. [ABSTRACT FROM AUTHOR]

The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only ∼11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, ∼16.34 ± 4.02% to 19.37 ± 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 ± 0.29% to 2.09 ± 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10–1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy. [ABSTRACT FROM AUTHOR]

Mammalian cell expression systems are the most commonly used platforms for producing biotherapeutic proteins. However, development of recombinant mammalian cell lines is often hindered by the unstable and variable transgene expression associated with random integration. We have developed an efficient strategy for site-specific integration of genes of interest (GOIs). This method enables rapid and precise insertion of a gene expression cassette at defined loci in mammalian cells, resulting in homogeneous transgene expression. We identified the Hipp11 (H11) gene as a "safe harbor" locus for gene knock-in in CHO-S cells. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated homologous recombination, we knocked in a DNA cassette (the landing pad) that includes a pair of PhiC31 bacteriophage attP sites and genes facilitating integrase-based GOI integration. A master cell line, with the landing pad inserted correctly in the H11 locus, was established. This master cell line was used for site-specific, irreversible recombination, catalyzed by PhiC31 integrase. Using this system, an integration efficiency of 97.7% was achieved with green fluorescent protein (GFP) after selection. The system was then further validated in HEK293T cells, using an analogous protocol to insert the GFP gene at the ROSA26 locus, resulting in 90.7% GFP-positive cells after selection. In comparison, random insertion yielded 0.68% and 1.32% GFP-positive cells in the CHO-S and HEK293T cells, respectively. Taken together, these findings demonstrated an accurate and effective protocol for generating recombinant cell lines to provide consistent protein production. Its likely broad applicability was illustrated here in two cell lines, CHO-S and HEK293T, using two different genomic loci as integration sites. Thus, the system is potentially valuable for biomanufacturing therapeutic proteins. [ABSTRACT FROM AUTHOR]

Background: The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. Methods: In order to approach the natural conditions of infection and possible integration, we generated linear JcDV-gfp based molecules which were transfected into non permissive Spodoptera frugiperda (Sf9) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. Results: We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear “scrambled” whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent downregulation by non-structural proteins of the VP-gene cassette promoter. Conclusion: Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences. [ABSTRACT FROM AUTHOR]

Abstract The intranasal route of administration allows large therapeutics to circumvent the blood–brain barrier and be delivered directly to the CNS. Here we examined the distribution and pattern of cellular transfection, and the time course of transgene expression, in the rat brain after intranasal delivery of plasmid DNA nanoparticles (NPs) encoding hGDNF fused with eGFP. Intranasal administration of these NPs resulted in transfection and transgene expression throughout the rat brain, as indicated by eGFP ELISA and eGFP-positive cell counts. Most of the transfected cells were abluminal and immediately adjacent to capillaries and are likely pericytes, consistent with their distribution by perivascular transport. Intranasal administration of these plasmid DNA NPs resulted in significant, long-term transgene expression in rat brain, with highest levels at 1 week and continued expression for 6 months. These results provide evidence in support of intranasal DNA NPs as a non-invasive, long-term gene therapy approach for various CNS disorders. Graphical Abstract Intranasal administration of plasmid DNA nanoparticles expressing hGDNF (pUGG NPs) results in transfection of perivascular cells in the brain. (1) Intranasally delivered pUGG NPs are distributed along the rostral–caudal axis of the brain in the perivascular spaces surrounding capillary endothelial cells (EC). (2) pUGG NPs transfect primarily perivascular cells that are located abluminal and immediately adjacent to capillaries. (3) These transfected perivascular cells, which are likely pericytes, express the transgene, i.e. the reporter protein eGFP. AC: astrocyte; EC: endothelial cell; eGFP: enhanced green fluorescent protein; M: microglia; N: neuron; PC: pericyte or perivascular macrophage; PVS: perivascular space. Unlabelled Image [ABSTRACT FROM AUTHOR]

Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression. [ABSTRACT FROM AUTHOR]

Mougeotia scalaris is a filamentous streptophyte alga renowned for light‐inducible plastid rotation and microtubule‐dependent polarity establishment. As a first step toward transgenic approaches we determined the 5,825 base pair genomic sequence encoding the α‐tubulin1 gene (MsTUA1) of M. scalaris (strain SAG 164.80). The subcloned MsTUA1 promoter facilitated strong transgene expression in M. scalaris and tobacco leaf cells, as shown by particle bombardment and the subsequent visualization of expressed fluorescent protein markers. Our results provide a route for the genetic transformation of the filamentous streptophyte alga M. scalaris based on the endogenous TUA1 promoter. [ABSTRACT FROM AUTHOR]

Highlights • An automated method of quantifying motor axon labeling in nerve sections is proposed. • This method uses ImageJ and triple labeling of peripheral nerve sections. • This method is faster and has less variation than manual and semiautomated methods. Abstract Background Determination of transgene expression in motor axons of peripheral nerves is important in evaluating the effectiveness of viral transduction. Currently only manual and semi-automatic methods of quantification have been employed for quantification in immunolabeled nerve sections, but automatic methods exist for axon counting only in brightfield sections. Manual and semi-automatic methods can suffer from inter- and intraobserver bias, sampling bias and can be time consuming to implement. New Method A fully automated method using ImageJ and the Nucleus Counter plugin was developed to quantify the fraction of green fluorescent protein (GFP) labeled acetylcholine transferase positive axons in triple immunolabeled peripheral nerve sections. This method utilizes the Nucleus Counter to generate axonal regions of interest which are quantified for colocalization with GFP expression and nonoverlap with Laminin. Thresholding using histograms generated from control animals is used to remove noise. Results The automated method is able to successfully distinguish transgenic GFP expressing mice from wild type. Using computer generated peripheral nerve sections, the automated method has less than 5% error at signal-to-noise ratios greater than 10% of baseline. Comparisons with Existing Methods This method has comparable performance in false positive rates (<1%) and a 95% predictive interval that closely matches existing fully automated methods for quantification in brightfield sections. It outperforms the intra- and interobserver differences of manual and semi-automated methods for quantification. Conclusions This automated quantification method provides a fast and robust means of determining the fraction of labeled axons in peripheral nerve sections. [ABSTRACT FROM AUTHOR]